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. Spacers not only ensure that the distance between the nonamer and heptamer is correct but they also regulate recombination activity by providing protein-binding sites along the DNA sequences that affect recombination 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 . V(D)J rRSSs 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 . The hepDespite 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  and are Our current knowledge about the functional role of the spacer is that its length is crucial in directing V(D)J recombination . ComprehOne 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 , Jβ2.6 iApaLI 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 . 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 , top wasment see , which rment see .Recombination of Jβ2.6 RSS is below the level of detection of our assay . SubstitIn 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 , alone or in combination with a consensus heptamer and/or spacer, partially reproduces the effects of the full consensus nonamer A. IntereIn 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 , containTwo 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 A. The liInterestingly, a favorable spacer sequence (Sk or Sc), when paired with H or N, boosts cleavage over the Jβ2.6 RSS spacer B and 4D.It is likely that differences in the nucleotide sequences of the RSS lead to variations in the stability of RAG–RSS complexes . This idIn 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 A and 5B.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.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).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 J recombination has recently been developed , 2003. TSr = 0.90) than with binding (Sr = 0.86) and most correlated with SJ formation (Sr = 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  that were previously thought to be unimportant.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 . 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.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 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  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 .Hin recombinase . The Jβ2.6 RSS nonamer differs from the consensus in the second and fourth positions see , and theombinase  suggest ombinase . Hence, ombinase . Having ombinase ; while pPrevious 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  . In contThe effect of a consensus spacer on 12-SC formation exhibits striking plasticity see . AdditioOur 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 , it is cFinally, 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 . Spacer Based on comprehensive sequence alignments showing a small but significant degree of spacer sequence conservation , a few sPpoI and I-CreI homing endonucleases, which cleave at recognition sites approximately 20 bp in length  interactions of a strictly sequence-specific nature, and “analog” (or multiplicative) contacts that fine-tune the strength of the digital contacts . Digitalmax, where A = effect on binding efficiency due to analog interactions) to no activity (0 × Amin). Hence, the noncritical residues are crucial for determining how well a protein complex can exert its biological function.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 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  versus true binding sites whose sequences diverge markedly from the consensus .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  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 , 2003. FINNE1, CIT4A, TL1, TL2, TL3, TL4, TL5, and TL6) have been described previously , and the double-stranded fragment was digested with HindIII and SalI 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.To generate the library for the genetic screen, the oligonucleotide pEBB, pEBB-RAG1, and pEBB-RAG2 expression constructs have been described elsewhere .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 . To detect SJs, DNA samples were treated with DpnI, MluI, and XhoI to remove unreplicated and unrecombined plasmids. Oligonucleotides INNE1 and CIT4A were used to amplify SJs . To detect CJs, RAP samples were treated with DpnI 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 , and quantified using ImageQuant software (Molecular Dynamics).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 ; for conDpnI/MluI/XhoI (for cloning of SJs) or DpnI 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.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 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.Recombinant GST-RAG2, MBP-RAG1, and HMG2 were expressed and purified as described previously .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 .Click here for additional data file.Table S1(31 KB DOC).Click here for additional data file.Table S2(23 KB DOC).Click here for additional data file.


Since LPS is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. Although LPS has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium.In animal pathogenic bacteria, horizontal gene transfer events (HGT) have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide (LPS). As a result, different strains of the same pathogen can have substantially different Xanthomonas oryzae pv. oryzae (Xoo) encoding six genes whose products are homologous to functions involved in LPS biosynthesis and transport. All six open reading frames (ORFs) have atypical G+C content and altered codon usage, which are the hallmarks of genomic islands that are acquired by horizontal gene transfer. The lps locus is flanked by highly conserved genes, metB and etfA, respectively encoding cystathionine gamma lyase and electron transport flavoprotein. Interestingly, two different sets of lps genes are present at this locus in the plant pathogens, Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas axonopodis pv. citri (Xac). The genomic island is present in a number of Xoo strains from India and other Asian countries but is not present in two strains, one from India (BXO8) and another from Nepal  as well as the closely related rice pathogen, Xanthomonas oryzae pv. oryzicola (Xoor). TAIL-PCR analysis indicates that sequences related to Xac are present at the lps locus in both BXO8 and Nepal624. The Xoor strain has a hybrid lps gene cluster, with sequences at the metB and etfA ends, being most closely related to sequences from Xac and the tomato pathogen, Pseudomonas syringae pv. tomato respectively.We report here the complete sequence of a 12.2 kb virulence locus of lps locus between different strains of a plant pathogenic bacterium. Our results indicate that multiple HGT events have occurred at this locus in the xanthomonad group of plant pathogens.This is the first report of hypervariation at an  Pseudomonas aeruginosa, an opportunistic human pathogen [Vibrio cholerae is considered as a major cause for the cholera epidemic that originated in India in 1992 [lps gene repertoire is to be expected within different strains of plant pathogenic bacteria.LPS is an important constituent of the outer membrane of gram-negative bacteria. Variation in LPS composition can have profound consequences for these cells by potentially providing resistance against bacteriophages and antimicrobial compounds as well as facilitating evasion of the host immune system in animal pathogens. Extreme variation at LPS gene clusters has been reported in animal pathogenic bacteria. Recently, eleven highly divergent gene clusters were reported to occupy an LPSspecific locus in pathogen . The acq in 1992 -8. LPS h in 1992  and in s in 1992 ,12 as we in 1992 . Since LXanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoor) cause diseases of rice [The genus Xathomonas includes a number of plant pathogenic bacteria. Two related members of this genus,  of rice . They ex of rice -19. In IwxoD, wzt and wzm, that are postulated to be involved in LPS biosynthesis and transport. These newly described genes also have atypical G+C content and all the six genes at this locus exhibit altered codon usage pattern, as compared to other Xoo genes. We present evidence that this locus is present in many, but not all, Xoo strains and that it is absent in Xoor. Our results indicate that there is substantial variation at this locus among various xanthomonads. The possible significance of these results is discussed.In previous research, we have reported a 5.5 kb region in the genome of Xoo strain BXO1 and demonstrated that it encodes three genes that are involved in biosynthesis of LPS and extracellular polysaccharide (EPS) as well as in virulence . All theEcoRI fragments . We subcloned all the fragments into pBlueScript. Based on the end sequences of the inserts in the subclones and pSD5, the lps locus was mapped to four of these EcoRI fragments . The previously obtained sequence was found to include all of the 3.5 kb and part of the 4 kb fragment and the remaining sequence of this region was obtained by sequencing the 0.6 kb and the 9 kb fragment (Please refer Methods). A total sequence of 13.18 kb was constituted by joining 6.14 kb of previously obtained sequence [lps locus and some flanking regions. The additional sequence of the lps locus encodes three putative genes which encode a predicted O-antigen acetylase, a predicted ABC transporter permease and a predicted ATP-binding protein and three insertion sequence (IS) elements.In an earlier study, a novel Xoo locus was reported to be required for LPS and extracellular polysaccharide (EPS) production as well as virulence. A 35 kb cosmid, pSD5, that complements mutations in this region was isolated . PartialwxoA (encodes a predicted epimerase), wxoB and wxoC (both encode predicted glycosyl transferases) have been described earlier. The fourth gene is wxoD and encodes a predicted 327 amino acids long protein. A BLAST [Mesorhizobium loti . Interestingly, no homologs of this gene have been reported in any other xanthomonad. The fifth gene, wzt, encodes a predicted 436 amino acid long protein. A BLAST search reveals homology to functions involved in LPS transport. The best match is with the ATPase component of an ABC-type polysaccharide transport system from Burkholderia fungorum . The sixth gene, wzm, encodes a predicted 437 amino acid long protein which is homologous to integral membrane protein components of ABC transporter systems that are involved in LPS transport. The best match is with a permease component of the ABC-type polysaccharide export system from Pseudomonas fluorescens PfO-1 . The start codon of wzt overlaps with the stop codon of wzm. Homologs of wzt and wzm are typically present in many lps gene clusters. Interestingly, two complete Insertion Sequence (IS) elements (ISXo8 and IS1113) and one truncated IS element (IS1114) interrupt this cluster between the genes, wxoD and wzt. ISXo8 is a novel 1320 bp long insertion sequence and a BLAST search shows homology to transposase of ISRSO17 encoded by Ralstonia solanacearum . A complete copy of the IS1113 element (AF482989) and a truncated copy of the IS1114 element (AF232058) are also present as indicated in Fig. lps loci [wxoA, wxoB, wxoC and wxoD might constitute one operon and that ORFs wzm and wzt might be transcribed together. The overlap between the start codon of wzt and the stop codon of wzm also suggests that these two genes are co-transcribed.All of the putative genes have been named as per Bacterial Polysaccharide Genes Nomenclature (BPGN) . The fir A BLAST  search rlps loci . Transcrlps locus is flanked by metB, which encodes a predicted cystathionine gamma lyase, and etfA which encodes a predicted electron transport flavoprotein. The genome sequences of Xanthomonas campestris pv. campestris  and Xanthomonas axonopodis pv. citri  have been obtained [metB gene  exhibits within the sequenced region, 91% and 88% nucleotide identity to metB genes of Xac (AE012010.1) and Xcc (AE012157.1), respectively. The Xoo etfA gene  exhibits within the sequenced region, 93% and 91% nucleotide sequence identity, respectively, with etfA genes in Xac (AE012009.1) and Xcc (AE012159.1). Interestingly, the lps biosynthetic gene cluster of Xcc, which comprises fifteen genes, is also located between the metB and etfA genes [The obtained . The XoofA genes . In Xac,lps locus is 50.46% (excluding the IS elements) [Fig. wxoD) to 56.3% (wxoC). Atypical G+C content is a characteristic feature of "genomic islands" that are believed to be acquired by horizontal gene transfer. The transposase genes encoded by ISXo8 and IS1113 have a G+C content that is >61%, a value which is typical for the genomes of Xoo and other xanthomonads. The G+C content of metB and etfA genes that flank the genomic island have G+C content of 64.3% and 61% respectively  which is typical of the Xoo genome.The average G+C content of Xoo and other Xanthomonads is estimated to be around 65% , while tts) Fig. . The varCodon Usage Pattern or CUP (Please refer Methods). Eight aminoacids, i.e., Glycine, Valine, Threonine, Leucine, Arginine, Serine, Proline and Alanine, were selected to study CUP because they have atleast four synonymous codons. The percentage of synonymous codons that end with G or C was calculated for each aminoacid and gene. This analysis was conducted for six genes of the lps island and six genes from elsewhere in the Xoo genome (please refer Methods). We show that CUP of the genes present in the genomic island is dramatically different from the typical Xoo genes  of Xoo strains that we have examined from India , and different sized bands in BXO8 (lane 3), Nepal624 (lane 4) and BXOR1 (lane 2) indicating that the metB gene is present but located in different EcoRI fragments  PCR is an efficient technique for isolation of target DNA segments adjacent to known sequences [metB and etfA genes suggests that sequences which are significantly similar to the Xac lps gene cluster are present at this genomic location in both of these strains. Next to metB, a wzm homolog is present in BXO8  and Xac with 69.2% identity at nucleotide level within the sequenced region. Next to etfA, a putative integral membrane protein encoding gene is present in both BXO8  and Xac with 91.3% identity at nucleotide level within the sequenced region. The BXO8 and Nepal624 strains exhibit 100% nucleotide sequence identity within the sequenced region. TAIL- PCR analysis of the Xoor strain indicates that it has a hybrid lps gene cluster. Next to metB, a unique wzm gene is located  which exhibits 62.8% nucleotide identity to wzm gene of Pseudomonas syringae pv. tomato strain DC3000 (AE016859.1). Next to etfA, a putative inner membrane protein encoding gene is located  which exhibits 97% and 92% nucleotide sequence identity, respectively, with similarly located genes in BXO8 and Xac. Because the BXO8 and Nepal624 strains have different sequences at the lps locus, as compared to other Xoo strains, we inoculated these strains along with appropriate controls onto leaves of the susceptible rice cultivar Taichung Native-1. We find that BXO8 and Nepal624 strains are able to cause typical bacterial leaf blight disease symptoms that are indistinguishable from those elicited by other Xoo strains (data are not shown).What are the sequences present at this genomic location in the Xoo strains that lack the equences . TAIL-PCmetB and etfA genes in BXO1 and BXO8. The homology breakpoints appear to localise to the 3' regions of metB and etfA genes, exactly 18 bp upstream of their respective stop codons. Upto the break points, within the sequenced region at either end of the lps locus, the nucleotide sequence is identical in BXO1, BXO8 and Nepal624. The DNA sequence immediately preceding the break points was examined manually for presence of direct or inverse repeats. Interestingly, we could find three inverted repeats  within the 3' regions of metB and etfA near the homology breakpoints between BXO1 and BXO8  flanking the lipopolysaccharide gene cluster has been reported in Mycobacterium avium subsp. paratuberculosis [Escherichia coli strain K5, Vibrio cholera, etc. This sequence was implicated to be involved in gene regulation and has also been suggested to have a role in recombination [The presence of invert repeats in the regions that flank the rculosis . Anotherbination ,36.lps loci of animal pathogenic bacteria are under intense host selection and extreme variation is reported in lps specific gene clusters [lps gene clusters suggests that the plant pathogenic bacteria are also under selection to vary their LPS. Alterations in LPS composition might result in resistance against predators like bacteriophages [As LPS is highly immunogenic, clusters . The obsiophages ,10 or reiophages  in the hlps biosynthetic gene clusters within different strains of a plant pathogenic bacterium. The results also indicate that multiple HGT events have occurred at this locus in various xanthomonads and provide a new parallel in the mechanisms that plant and animal pathogenic bacteria can employ to generate variability in cell surface molecules.These results provide, for the first time, evidence for substantial variation in lps locus was cloned as part of a 35 kb cosmid clone, pSD5. The insert includes 0.6, 2.2, 3.5, 4.0, 6.0, 9.0 and 10 kb fragments upon EcoRI  digestion and all the fragments were subcloned in to pBlueScript . Most of the sequence obtained in this study was generated by sequencing the 9 kb subclone, pBP4, using a modified shotgun sequencing procedure. Here, pBP4 was digested with EcoRI and the 9 kb fragment was gel eluted. Then the fragment was partially digested (1.5–2.5 kb) using a blunt-end cutter, HaeIII (NEB) and cloned into pMOS . The inserts were amplified from random clones by colony PCR using vector primers and were sequenced using an ABI Prism 3700 automated DNA sequencer . After editing, the assembly of the sequence data was done using GeneTools  and Blast2 [lps locus that was obtained using long range PCR  with BXO1 genomic DNA as template. The sizes of the fragments corresponded to the sizes that are predicted by in silico analysis of the sequence (data are not shown). The ORF's were assigned using ORF finder [EcoRI fragment which is also a part of the lps locus. The Pbp1 primer binds just after the wxoC ORF (which forms part of the 3.5 kb EcoRI fragment) and Pbp2 binds within the wxoD ORF (which forms part of the 9.0 kb EcoRI fragment). A 0.67 kb PCR amplified fragment is obtained from BXO1 genomic DNA using Pbp1 and Pbp2. The band was gel eluted and was sequenced using Pbp1 and Pbp2. The sequence was found to include the 0.6 kb EcoRI fragment. In addition, the sequences of all six ORFs were confirmed by sequencing of PCR amplified fragments from genomic DNA using specific sets of gene specific primers (see the list of primers in Table The d Blast2 . MultiplF finder . Two prilps locus. These genes encode: a putative siderophore receptor (AF325732), Xanthomonas adhesin like protein (AF288222), a putative phytase (AY151260), rpfF (AF411962), shikimate dehydrogenase (AF258797) and secreted xylanase (AF331922). The second group comprised the six genes (excluding transposases) encoded in the Xoo lps gene cluster (AF337647).For each gene the frequency of codon usage for different aminoacids was calculated using a web based program . FurtherEcoRI (NEB) according to supplier's instructions. Digested genomic DNA was separated on a 0.8% agarose gel and vacuum transferred to a Hybond N+ filter (Amersham) using 0.4% NaOH as described by Sambrook et al. [32P dATP using random primer labelling kit . Prehybridization, hybridisation and washings were done at 68°C as described by Yashitola et al [Specific oligonucleotide primer pairs were designed and used to amplify gene specific fragments for each of the ORFs encoded in the BXO1 genomic island  was lysed in 100λ of 0.01 N NaOH by boiling for 10 minutes. After spinning at 13 K for 1 min., 2λ of supernatant was used as template for PCR using the gene specific primers described above. The products were separated by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining.To screen for the presence of the genomic island in different strains, a procedure for colony PCR was standardized. A portion of a single colony ; Sequences of TAIL-PCR products from metB end of BXO8 (AY319936), Nepal624 (AY319938) and BXORI (AY319940); Sequences of TAIL-PCR products from etfA end of BXO8 (AY319937), Nepal624 (AY319939) and BXORI (AY319941).The nucleotide sequences obtained in this study have been deposited in GenBank with the following Accession numbers: Sequence of PBP carried out all the aspects of the work and drafted the manuscript. RVS conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Most of the existing RNA structure prediction programs fold a completely synthesized RNA molecule. However, within the cell, RNA molecules emerge sequentially during the directed process of transcription. Dedicated experiments with individual RNA molecules have shown that RNA folds while it is being transcribed and that its correct folding can also depend on the proper speed of transcription.The main aim of this work is to study if and how co-transcriptional folding is encoded within the primary and secondary structure of RNA genes. In order to achieve this, we study the known primary and secondary structures of a comprehensive data set of 361 RNA genes as well as a set of 48 RNA sequences that are known to differ from the originally transcribed sequence units. We detect co-transcriptional folding by defining two measures of directedness which quantify the extend of asymmetry between alternative helices that lie 5' and those that lie 3' of the known helices with which they compete.We show with statistical significance that co-transcriptional folding strongly influences RNA sequences in two ways: (1) alternative helices that would compete with the formation of the functional structure during co-transcriptional folding are suppressed and (2) the formation of transient structures which may serve as guidelines for the co-transcriptional folding pathway is encouraged.These findings have a number of implications for RNA secondary structure prediction methods and the detection of RNA genes. However, theoretical studies of RNA molecules [functional structure which confers the desired functionality within the organism to the RNA molecule.Most of the existing computational methods for RNA secondary structure prediction fold an already completely synthesized RNA molecule. This is done either by minimizing its free energy  which target the emerging RNA or whether the primary structure of the RNA molecule itself conveys the desired properties to guide its own correct co-transcriptional folding.In this paper, we propose several statistics in order to detect, if and how co-transcriptional folding influences RNA sequences. Using these statistics, we show that the effects of co-transcriptional folding are widespread in RNA genes.We want to show that an RNA sequence is organized in such a way to help the formation of the functional secondary structure during transcription. We aim to support this hypothesis by detecting two different features:Possible competitors of helices in the functional structure are suppressed. When the 3' end of a helix that is part of the final secondary structure emerges during transcription, the number of possible competitors for the 5' part of the helix should be as low as possible in order to promote the formation of the correct helix.• The folding pathway is engineered. During transcription, several temporary helices are formed which may guide the folding process.• H0 case, when we suppose no co-transcriptional folding. However, verifying that these statistics have an expectation value of zero in the H0 case cannot simply be achieved by analyzing random sequences. Indeed, even generating random sequences is not trivial. First, it is hard to reliably predict the minimum free energy structure for the randomized sequences as most secondary structure prediction algorithms discard pseudo-knots and, even without pseudo-knots, predict only on average about 70 % of the base-pairs correctly. In addition, there is no guarantee that the secondary structure with the lowest free energy would correspond to the functional one. Second, even if the random sequences are generated by a shuffling algorithm which keeps the given secondary structure fixed, it cannot be guaranteed that the fixed structure remains the correct one for the new primary sequence. Generating random sequences therefore provides no straightforward solution for obtaining a H0 statistics with expectation value zero.We investigate these features using several statistics which are based on the known primary and secondary structures of our RNA sequences. A crucial point in investigating these features is to define a set of statistics that have expectation of zero in the H0 case and where one statistics has a bias away from the H0 expectation value in case of co-transcriptional folding, while the other statistics is not affected by co-transcriptional folding. By studying the difference of these two statistics, we thus gain a new statistics with expectation value zero in the case of no co-transcriptional folding and an expectation value larger or smaller than zero in the case of co-transcriptional folding.We circumvent this problem by studying pairs of statistics, where both statistics have the same, unknown expectation value in the minstem = 9 consecutive base-pairs of type {G - C, C - G, A - U, U - A, G - U, U - G} and are calculated by a dynamic programming procedure in which the known primary and secondary structure of the RNA is fixed, see Figure minstem values (data not shown). While calculating all helices of at least minstem length, we test which of these helices constitute competing alternatives to helices of the known secondary structure and record each such competing case in one of our statistics. These alternative helices may be part of a pseudo-knotted structure and we do not discard them. As each of the two bases i and  of a base-pair in a known helix can have a competing alternative base-pairing partner within an alternative helix and as this alternative partner can either be found 5' (before), 3' (behind) or between the two strands of the known helix, all cases can be classified into six different classes. Of these six, we discard the two classes where the alternative helix falls between the two strands of the known helix as this un-paired loop region is typically too short to accommodate an alternative helix of at least minstem length. The remaining four classes, see Figure cis- and two trans- alternative classes, depending on whether the known base-pairing partners lie between the alternative base-pairing partners (trans) or not (cis). The four statistics 3'cis, 3'trans, 5'cis and 5'trans that we use correspond to these four classes.The statistics (which we will define in detail below) measure the presence of alternative helices which compete for at least one base-pair with the helices of the known secondary structure. These competing alternative helices are required to consists of at least cis- and trans-alternative helices are not necessarily the same as the stacking energies are not symmetric with respect to the 5' → 3' direction of the RNA sequence [cis-pairing partners are closer to the known pairing partners than trans-pairing partners and may thus lead more easily to incorrect helices. We may therefore compare only cis-competitors with other cis-competitors and trans-competitors with other trans-competitors. This yields two possible comparisons: 3'trans versus 5'trans and 5'cis versus 3'cis, see Figure It is important to note that even without co-transcriptional folding, the destabilizing effects of competing cis, 3'trans, 5'cis and 5'trans, where an alternative helix competes with a known helix. Each such event is given two different weights, see Table d·log(l)), where d is the distance between the two competing helices and l is the length of the sub-sequence 5' or 3' of the known helix on which the competing helix falls, or (2) a weight of |G| / (d·log(l)), where the former weight is multiplied by the absolute value of the free energy G of the competing, alternative helix. The factor 1/d gives alternative helices that are far away from the known helix a smaller weight than closer ones. The factor 1/log (l) accounts for the fact that log (l) is proportional to the expected sum of 1/d statistics for a sub-sequence of length l . The free energy factor G in the second type of weights gives stable alternative helices which have a larger impact on the folding pathway a greater weight than helices which are easily unfolded. Statistics derived from weights of type 1/(d log(l)) are denoted by an index p (for plain) and those of type |G| / (d·log(l)) by an index g (for free energy). By summing the weighted counts for each of the four classes of events, we thus arrive at eight different scalar values which characterize each RNA sequence: 3'Transx, 3'Cisx, 5'Transx and 5'Cisx for x ∈ {p,g}.We proceed as follows to detect if co-transcriptional folding takes place: For every RNA sequence of the data set, we detect events of type 3'We can now define the two statistics which are capable of measuring the two main types of asymmetry within each RNA sequence:Cis := 5'Cis - 3'CisTrans := 3'Trans - 5'Trans configurations, see Figure  configurations). Both types of effects are indicated by an expectation value larger than zero for the respective statistics.which can calculate for both types of weights. Without co-transcriptional folding, the expectation value of these two statistics is zero. Co-transcriptional folding induces two types of asymmetries by suppressing the number of alternative helices which compete with the final helices (indicated by an increased number of (pos) thus follows a binomial distribution with parameter p = 0.5 and the statisticWithout co-transcriptional folding, the introduced statistics have an expectation of zero, moreover, the distributions should be symmetric. The number of positive cases n is the number of all cases, approximately follows a standard normal distribution. If this value is sufficiently positive, we have to reject the hypothesis that co-transcriptional folding is not encoded within RNA genes.where All 16S rRNA, 23S rRNA as well as Group I and Group II type intron sequences with completely known secondary structures were downloaded from the Comparative RNA Web (CRW) Site ,36, resuOrganellar 23S rRNA sequences frequently contain Group I introns and recent research revealed that the 23S rRNA of several hyperthermophilic bacteria also have Group I intron . Other sVibrio harvey, the order is 23S-16S-5S [Vibrio cholerae [Vibrio parahaemolyticus [rRNA genes in bacteria are encoded in the so-called rrn-operon (see for example ). The caS-16S-5S , but notcholerae  and Vibrolyticus , whose 1We divided the gathered sequences into two sets: data set A which consists of all RNA sequences that are thought to correspond to the originally transcribed sequence units and data set B which contains all those RNA sequences that are known to differ from the originally transcribed sequence units. Data set B thus contains the Group I and II intron sequences, organellar and hyperthermophilic bacteria 23S RNA sequences. As we neither know the sequence nor the secondary structure of the original transcript units from which the sequences of data set B were derived, we are limited to detecting the effects of co-transcriptional folding within these shorter sequences. We expect this to be much more difficult than in sequences that correspond to the originally transcribed sequence units as co-transcriptional folding introduces long range effects which are harder to detect the shorter the investigated sub-sequence gets. See Table 3'Cisx, 3'Transx, 5'Cisx and 5'Transx values for both types of weights, i.e. x ∈ {p,g}, for each sequence in the two data sets. From these values we then derived each sequence's Cisx and Transx values, again for both x types of weights. Their distributions are shown in Figure We calculated the The first thing to note in Figure Cis and Trans in Table The mean values of Cis value larger zero means that configurations of type  outnumber those of type , see Figure c and i that can later yield to the final secondary structure element containing the base-pair between i and  thus seems to be encouraged. However, these transient structure elements may not be too stable if they are to guide rather than impede the proper folding. The presence of transient helices could thus be further substantiated by showing that these transient helices are less stable than the final helix. In contrast to the  configuration, the competing ic helices in the  case are suppressed as they lie 3' of the final  helix and thus emerge later in time during co-transcriptional folding. A Cis value larger than zero can therefore be explained by the presence of temporary helices which may guide the formation of the final, functional secondary structure during co-transcriptional folding.A Trans value larger than zero means that  configurations are less frequent than  configurations, see Figure  configuration, both c and  are competing pairing partners for i as they both emerge before i during transcription. This may lead to the formation of wrong ci helices, whereas the order of pairing partners in the  configuration has a lower risk of mis-folding due the c emerging only after the  and thus only after the  helix could have already formed.A 3'Trans > 3'Cis in Table  or  helix temporarily break up. In the case of the 3'Trans configuration, the pairing partners come in the order  along the RNA sequence, whereas they come in the order  in the 3'Cis configuration. In the  order, the c part is in vicinity to the i part, so the possibility of ending up with a wrong refolding due to a ic helix is larger than in the  case.In addition, (3'Trans > 3'Cis), that the formation of temporary helices may guide the structure formation and that these helices may thus be used to actively engineer a folding pathway (Cis >0) and that secondary structure elements which may interfere with the formation of the final, functional secondary structure during co-transcriptional folding are suppressed (Trans >0).Overall, we can thus conclude from the average values in Table In order to quantify the influence of co-transcriptional folding further, we calculated two statistics, a t-test for the hypothesis that the given statistics have an expectation value of zero as well as the p-value of the number of positive cases for our two co-transcriptional folding indicators, see Table Recent experimental studies ,24,19 haAlthough our statistics are able to reveal two general effects of co-transcriptional folding within data set A, we cannot conclude that they would be powerful enough to serve as a reliable indicator of co-transcriptional folding for single RNA sequences, as some of the sequences in data set A may not correspond to the originally transcribed sequence units. In addition, all of our statistics consider only a first order effect of co-transcriptional folding by studying alternative helices for the known helices, but do not take higher order effects into account as e.g. alternative helices of alternative helices etc.Based on computer simulations, H. Isambert et. al.  conjectuTo summarize, our findings show that co-transcriptional folding is a guiding principle in the formation of functional RNA structure and that it can influence both the primary and potential secondary structures of an RNA molecule. This has several implications. Current algorithms for RNA secondary structure prediction can probably be improved by adopting co-transcriptional folding as a guiding principle rather than only free energy minimization. This may hopefully provide the extra information needed to be able to reliably detect RNA genes . Severalhow RNA sequences fold. This should in turn enable us to also understand why some RNA sequences mis-fold and fail to function properly in the organism. Even though protein folding is known to differ in many respects from RNA folding, they also have some features in common [co-translational folding.Most importantly, co-transcriptional folding should lead to a better understanding of n common . One of In this study, we neither attempted to study the effects that co-transcriptional folding may have on sequences that are transcribed together (e.g. genes in an operon) nor to study the influence that the binding by proteins or RNA sequences or RNA editing may have on the co-transcriptional folding pathway and the final, functional RNA structure. This will almost certainly require more refined investigation methods, but we hope that this study provides enough insight and motivation to start to tackle these exciting questions.I.M.M. proposed this work and contributed the main idea for the statistics. I.M. selected the data and evaluated the statistical significance of the results. Both authors shared the programming tasks and the writing of the manuscript.


External and internal factors are increasingly encouraging research funding bodies to demonstrate the outcomes of their research. Traditional methods of assessing research are still important, but can be merged into broader multi-dimensional categorisations of research benefits. The onus has hitherto been on public sector funding bodies, but in the UK the role of medical charities in funding research is particularly important and the Arthritis Research Campaign, the leading medical charity in its field in the UK, commissioned a study to identify the outcomes from research that it funds. This article describes the methods to be used.A case study approach will enable narratives to be told, illuminating how research funded in the early 1990s was (or was not) translated into practice. Each study will be organised using a common structure, which, with careful selection of cases, should enable cross-case analysis to illustrate the strengths of different modes and categories of research. Three main interdependent methods will be used: documentary and literature review; semi-structured interviews; and bibliometric analysis. The evaluative framework for organising the studies was previously used for assessing the benefits from health services research. Here, it has been specifically amended for a medical charity that funds a wide range of research and is concerned to develop the careers of researchers. It was further refined in three pilot studies. The framework has two main elements. First, a multi-dimensional categorisation of benefits going from the knowledge produced in peer reviewed journal articles through to the health and potential economic gain. The second element is a logic model, which, with various stages, should provide a way of organising the studies. The stock of knowledge is important: much research, especially basic, will feed into it and influence further research rather than directly lead to health gains. The cross-case analysis will look for factors associated with outcomes.The pilots confirmed the applicability of the methods for a full study which should assist the Arthritis Research Campaign to demonstrate the outcomes from its funding, and provide it with evidence to inform its own policies. Health research funding bodies are under increasing pressure to demonstrate the outcomes, or benefits, of the research that they fund -6. TradiThe onus hitherto has been on public sector funding bodies. There is a general recognition in the UK, however, of the importance of the role of the medical charities: they fund approximately one third of UK medical research – a level 'unparalleled elsewhere in the world and nor is it found in other areas of science' . TherefoThe purpose of this paper is to set out the aims of the study and the methods being adopted. In particular, it will show how an existing generic approach to the assessment of benefits from health research ,12 has bAttention is being drawn to the increasing scale of the burden of musculoskeletal conditions, and the associated costs, in various ways including through the establishment of the Bone and Joint Decade 2000–2010 and the recent collaborative report with the World Health Organization (WHO) . At the ARC is the leading medical charity in this field in the UK and one of the largest collection-based medical charities in the UK. The Research Outputs Database (ROD) records the funding acknowledgements on all UK biomedical papers contained on the citations indices of the Institute for Scientific Information . AnalysiWithin the general climate of an increased emphasis on the outcomes from research, four main objectives were specified for the particular study described in this article:• Review and document outcomes for ARC research grants• Illustrate the strengths and weaknesses of different modes of research funding• Identify factors associated with translation of research, and attempt to develop 'early indicators' of likely successful translation• Identify 'good news stories' and vignettes of the research process for use by ARC in public engagement and fund raising activities.Traditional methods of peer review have long been favoured by medical research funding bodies for evaluating research, but bibliometric methods have had a variable history. An early move by the National Institutes of Health (NIH) to establish a publications' database was cancelled by the 1980s 'as too expensive for the management information it produced' . The MRCThe Economic and Social Research Council (ESRC) in the UK commissioned a project to identify the impact of their research on non-academic audiences. It involved tracing the activity of the participating researchers after their projects ended and mapping the networks of researchers and relevant non-academic users and potential beneficiaries . The stuCase studies will enable narratives or stories to be told to illuminate how the research funded in the early 1990s was translated (or not) into practice; each case, therefore, could potentially provide an illustrative example of the outcomes from ARC research. Furthermore, the planned 16 case studies will be based on a variety of modes of ARC research funding and types of research. They will also be organised using a common structure. This should enable cross-case study analysis to demonstrate (via illustrative case studies) the strengths and weaknesses of different modes of funding and categories of research. It should also facilitate the identification of factors associated with the translation of research, perhaps through various phases, into policies, products, and clinical practice that produce a health gain.The evaluation framework described below was developed in a way that incorporates previous experience and knowledge on these issues ,24,26,28In deciding the time window to use for selecting case studies, a compromise usually has to be made between the quality of records/likely ability of researchers to recall their activities and the selection of grants whose outputs have had sufficiently long to develop . The latWithin a case study approach it is unlikely that the selection of cases will follow a straight-forward sampling logic in which those selected are assumed to be representative of a larger group . NeverthAs a first step, we shall identify all publications in the relevant period from the principal investigators awarded ARC funds. Then, the researchers will be classified according to the journal impact factors (see below) of the journals in which their articles appear. The aim will be to draw up shortlists of possible researchers to include in the study: those in the top decile and those the middle of the range, with the final selection made on the basis of advice from ARC's Development Committee.For case studies it is appropriate to use multiple sources of evidence converging on the same issues  and adopWe will read key project documents including the original research grant proposals, referees' reports and end of project reports. On the basis of the end of year reports from researchers, and the interviews (see below), we will also identify and read the core publications attributed to the research grant and any subsequent publications such as key citing papers, relevant clinical guidelines etc.There will be about three interviews per case study. They will be based on a semi-structured interview schedule informed by the evaluation framework described below. They will, therefore, explore the origins of the research and the primary outputs such as the publications. In this way the initial list of publications identified as being related to the project will be refined. Furthermore, there will be a full exploration not only of the contribution to research training and career development, but also of any translation of the research findings into product development, policy and practice. In each case study the initial interviews will be with members of the relevant research team. Then snowballing techniques will be used to identify the people who might be able to provide most information about how the research has influenced subsequent research or been translated into product development, policy and practice.Bibliometric approaches can play a useful role in the analysis of the research funded by specific biomedical research-funding bodies ,31. In tIn every case a draft copy of the case study report will be sent to the principal investigator for comment. Such a step is an important part of the validation process and not just a matter of professional courtesy .There are two elements in the evaluation framework adopted to organise the case studies being conducted in the assessment of the outcomes from ARC-funded research. Building on the framework developed by Buxton and Hanney ,12, the There are many steps involved in assessing outcomes from research. One of the key advantages in taking a detailed approach, such as that described below, is that it enables the issue of the counter-factual to be addressed. In other words, what would the world have looked like without the specific research being examined?The multi-dimensional category of payback provides the evaluation criteria for the outputs and outcomes from ARC funding. The 5 main categories are:a) Knowledge productionb) Research targeting, capacity building and absorptionc) Informing policy and product developmentd) Health benefitse) Broader economic benefits.Each can be considered in turn, with various sub-categories explored and possible measures described.The knowledge produced by research is the first output and is contained in various publications and patent applications. Any type of publication can be considered, but it is generally thought that peer reviewed articles are the most important and, at least for biomedical research in industrialised countries, it is thought reasonable to assume that the overall output of research publications is fairly represented by peer-reviewed papers in international journals . In addiCitation analysis can be applied to assess the impact the specific article is having within the research community ,34. PrevA journal's 'impact factor' is based on the average number of times an article in the journal is cited; it can provide a short-hand version of citation analysis by giving some indication of the importance of the journal in which an article appears. The use of impact factors in analysis of biomedical research has been criticised  but, proParticularly when considering research that might be aimed at potential users outside the research community, it is often desirable to use a range of publication outlets including those journals with the highest readership among the groups at whom the research is targeted. In some fields these might well be journals that do not have an impact factor but are, nevertheless, significant as vehicles for dissemination of the knowledge produced -40.The better targeting of future research is frequently a key benefit from research, especially from research that is more basic and/or methodologically oriented. An indication of this comes from citation analysis. The enhanced targeting can be of the research conducted both by others and by the original researcher(s). Where follow-on research, especially by members of the original research team, is clearly associated with the original research it can be useful to obtain information on the source and amount of such funding . As is dResearch training can be provided both as a result of the employment of staff on research projects and programmes, and through explicit funding for research training and career development . One meaResearch can be used to inform policymaking in a wide range of circumstances and the key issue is that policymaking involves those in positions of authority making choices that have a special status within the group to which they apply . PolicymThe position of systematic reviews is a little complex. They are themselves a form of research, but inclusion of a study in a systematic review is a form of secondary output and might lead on to further use.At a similar level, although involving very different processes, research can also be used to inform product development . InformiBenefits in terms of health gains might be viewed as the 'real' payback or outcomes from health research. Greater effectiveness of health-care resulting from research-informed drugs or procedures should lead to increased health. Various measures of health gain exist, but for arthritis the emphasis, in most cases, is likely to be on those that assess reduction in pain or disability, and increase in mobility. While the benefits from arthritis research will not generally be measured in terms of life years gained, in some circumstances they might be captured by using Quality Adjusted Life Years (QALYs). This is often seen, in countries such as the UK, as a more appropriate approach than using Disability Adjusted Life Years (DALYs) . There hThis category of benefits can be thought of as going wider than health gain, and some aspects can be seen as benefits to the health sector more generally. Cost savings in the provision of health-care may result from research-informed changes in the organisation of services or in the particular therapies delivered. It might be necessary to consider various issues here. These include whether potential savings have in practice been realised – either as cash savings or as the release of resources for other valuable uses . FurtherA range of benefits can accrue to the national economy from the commercial exploitation of research. These can take the form of employment and profits resulting from the manufacture and sale of drugs and devices . The natWhilst there is a danger of double counting, it is probably also important to adopt a human capital approach and focus on the value of production gained from having a healthy workforce. This can be measured by examining the reduction in days off work. Typically, in a human capital approach, potential future earnings are calculated for people who, as a result of advances in medical research, can continue to contribute to national production ,15,48. TThe second element of the evaluation framework is the logic model. Its various stages are shown on Figure Stage 0: Topic/issue identificationInterface A: Project specification and selectionStage 1: Inputs to researchStage 2: Research processesStage 3: Primary outputs from researchInterface B: DisseminationStage 4: Secondary outputs – policymaking and product developmentStage 5: Adoption by practitioners and publicStage 6: Final outcomesWhile it is not possible totally to tie the categories of benefits to certain stages of the model, it is possible to identify broad correlations: categories a) and b) (knowledge and research benefits respectively) are together considered to be the primary outputs from research; category c) (informing policy and product development) relates to the secondary outputs; and categories d) and e)  are the final outcomes. This approach can be incorporated into the analysis of each stage in turn as is set out below, where a few examples, drawn from the pilot studies, are used to illustrate how the framework seemed to be working in practice but could be refined in certain ways.The topic or issue identification stage covers the generation of the original ideas for the research. Its nature can vary considerably depending on whether the main driving force is internally generated by the researcher, or externally generated . Most ARThe nature of the activities at Interface A will vary depending on the type of issue identification. Where the topics are externally generated, there are potential difficulties in ensuring both that the research community is actively engaged with the priorities that have been identified and that the project specification meets the needs as identified . Where tIt can be important to consider not only the financial inputs, including any beyond the specific ARC funding, but also the experience of the research team and the knowledge base on which they built. Part of the idea behind examining any other funding brought in to support ARC research is again to see how far ARC funding is helping to facilitate the funding of arthritis research by general funders of health research: is ARC funding studies that produce findings that others believe are worth further investigation? The pilot studies confirmed that the complexities of identifying the exact funding streams behind any piece of research were best addressed by using a case study approach involving initial documentary review and following up issues in interviews. The pilots involved a case where other contributory funding contributed to what was clearly an ARC project, and therefore little attempt was made to portion out credit for outcomes to any funder other than ARC. In another case, however, the research was part of a stream of ARC-funded work and an effort was made to try to draw boundaries around what would be appropriate to include in the case study.Consideration can be given to how appropriate the proposed methods for a study turned out to be, and whether any difficulties were encountered. In some cases it could be relevant to explore how far potential users were involved at this stage. It is possible that difficulties identified at this stage could explain later problems with translation or uptake of the research findings.Knowledge production, as represented by the various types of publications, is a major primary output from the research. Various ways of measuring this were discussed above. The pilots also showed that the interviews used to refine the lists of publications from the specific funding in question, could also sometimes help to identify where non-conventional sources were being used as outlets for publications. Most of the primary outputs will feed into the stock of knowledge.The research benefits in terms of targeting future research represent either feedbacks to further research conducted by team members, or findings that feed into the stock of knowledge and help target future research of others. An example from one pilot study showed not only how the principal investigator used her project to inform her own further work, but was also able to contribute to a much larger collaborative project. Interviews in another study showed that the research had informed considerable further work in industry, but as yet this had not led to any product development. Under the framework being used, it is possible to give that ARC-funded work considerable credit for informing the further research, but record its limited impact at the subsequent stages.Capacity building can also be seen as a primary output. Accounts were given, in pilot study interviews, of the research training and higher degrees that resulted from the research.Dissemination is usually seen as being somewhat more active than the mere production of academic publications containing the knowledge. There are, however, clear overlaps between some activities. Sometimes it is possible to record not just dissemination activities but also the successful transfer of research findings to potential users in the political, industrial, professional environment and wider society. Previous analyses of how to increase the implementation of research findings  will helAs noted above, policymaking and product development activities can result in a wide range of secondary outputs, and various methods are needed to identify research-informed policies. In one case study, a review of a database revealed that one project had been cited in a clinical guideline unbeknown to the research team, whereas in another pilot it took interviews to identify that the research was informing local guidelines and care pathways. The use of the research in systematic reviews was also revealed in various ways in the pilot studies. Where the research seems to have resulted in secondary outputs it is useful to explore the factors that have led to this.In relation to product development, if research findings are incorporated into the process of developing a product, for example a new drug for arthritis, this can be seen as an important secondary output. In the preliminary set of interviews, most people referred to how ARC-funded research had played a key role in the production of anti-TNF therapy for arthritis. In a pilot study, interviews revealed the extent to which industry's attempts to use one stream of research for product development had not, so far, been successful.For the research findings incorporated into secondary outputs to result in final outcomes there usually has to be some behavioural change by practitioners, and/or the public. This may involve take-up of new drugs or procedures as set out in a secondary output such as a guideline from the National Institute for Clinical Excellence (NICE). Sometimes the adoption comes as a direct result of the primary outputs, as when clinicians – often at the cutting edge – decide to implement research findings even prior to the development of clinical guidelines. Either way, it is important to try to establish the adoption or take-up rates and to explore how far the behavioural change can be attributed to the specific research findings, as opposed to other factors such as a more general change in climate of opinion in relation to, for example, the importance of exercise. In one pilot study where interventions based on research filtered into practice, a series of interviews was used to attempt to identify both the precise role of the specific ARC-funded project and possible levels of uptake.The role of the public in responding to informed advice – often research-based – is seen as increasingly important, especially in a field such as arthritis . VariousThe final outcomes are the health and broader economic benefits identified in categories d) and e) above. These are increasingly seen as being the ultimate goal of health research funding, but their precise estimate in practice often remains difficult -7. In onEach of the 16 cases will be written up as a narrative organised according to a common structure based on the various stages of the logic model. Each study should potentially, therefore, provide illumination as to the processes that could lead to outcomes and illustrations of such outcomes. In addition, the common structure of each case should facilitate some cross-case analysis that will not only look for common factors associated with research that has led to outcomes, but also see how far such outcomes are associated with different modes of funding and types of research. Some of this analysis should be based on the previous findings that are embedded into the evaluation framework: for example, basic research might be expected to produce a reasonable number of knowledge outputs but be less likely than clinical or AHP research to inform policies. Some other aspects of the analysis, however, are likely to be exploratory: detailed analysis of factors related to the role of medical charity research in contributing to outcomes appear, as yet, not to be well established.This paper sets out the aims and methods to be adopted in an innovative study to review the outcomes of the research funded by the Arthritis Research Campaign, one of the leading medical charities in the UK. At a time of growing emphasis on both accountability and evidence-based policy making, it is important for research-funding bodies to be able to show the results of their funding and base their policies on analyses of the processes involved in producing outcomes . Based oFinally, one of the challenges for the future will be to operationalise such analysis on a regular, and therefore less resource intensive, manner. It is hoped that the study will also shed light on these practical considerations, and do so in a way that will enable a system to be developed that meets the specific needs of the particular research funding body , in thisThe work was funded by ARC.JG led the design of the study, with all authors making contributions. SH drafted the article with contributions from all authors.


MRI slices of 1.5 mm thickness have been used in both cross sectional and longitudinal studies of osteoarthritis, but is difficult to apply to large studies as most techniques used in measuring knee cartilage volumes require substantial post-image processing. The aim of this study was to determine the optimal sampling of 1.5 mm thick slices of MRI scans to estimate knee cartilage volume in males and females for cross-sectional and longitudinal studies.A total of 150 subjects had a sagittal T1-weighted fat-suppressed MRI scan of the right knee at a partition thickness of 1.5 mm to determine their cartilage volume. Fifty subjects had both baseline and 2-year follow up MRI scans. Lateral, medial tibial and patellar cartilage volumes were calculated with different samples from 1.5 mm thick slices by extracting one in two, one in three, and one in four to compare to cartilage volume and its rate of change. Agreement was assessed by means of intraclass correlation coefficient (ICC) and Bland & Altman plots.Compared to the whole sample of 1.5 mm thick slices, measuring every second to fourth slice led to very little under or over estimation in cartilage volume and its annual change. At all sites and subgroups, measuring every second slice had less than 1% mean difference in cartilage volume and its annual rate of change with all ICCs ≥ 0.98.Sampling alternate 1.5 mm thick MRI slices is sufficient for knee cartilage volume measurement in cross-sectional and longitudinal epidemiological studies with little increase in measurement error. This approach will lead to a substantial decrease in post-scan processing time. Osteoarthritis (OA) is the most common form of arthritis and a leading cause of musculoskeletal disability in most developed countries . The kneThe present study consisted of two datasets; one was part of the Tasmanian Older Adult Cohort Study (TASOAC), an ongoing prospective population-based study aimed at identifying the environmental, genetic, and biochemical factors associated with the development and progression of OA at multiple sites , which commenced in 2002. Subjects aged between 50 and 79 years were selected randomly from the electoral roll of Southern Tasmania, with an equal number of males and females. Another dataset was a younger adult sample from the Knee Cartilage Volume study (KCV) as previously reported . Both stAn MRI scan of the right knee was performed on all subjects. Knee cartilage volume was determined by means of image processing on an independent work station using the software program Osiris as previously described ,15. Two Then, the volumes of all individual cartilage plates were recalculated based on different sampling intervals from 1.5 mm thick slices by extracting one in two, one in three, and one in four slice areas from the individual data file. These were then summed and the total was multiplied by the corresponding slice distance.Femoral cartilage volume was not assessed in this study as it is strongly correlated with tibial cartilage volume and thus adds little extra information , tibial 2).Weight was measured to the nearest 0.1 kg  using a single pair of electronic scales (Seca Delta Model 707) which were calibrated using a known weight at the beginning of each clinic. Height was measured to the nearest 0.1 cm (with shoes and socks removed) using a stadiometer. Body Mass Index (BMI) was calculated as weight (kg) / height  were from the TASOAC study and 50 subjects with longitudinal data  were from the KCV study. Characteristics of the study sample are presented in Table In cross-sectional analysis, compared to the cartilage volume measured using 1.5 mm thickness, decreasing the number of the slices by extracting one in two to one in four led to a very little underestimation in the magnitude of the average cartilage volume at lateral, medial tibial and patellar sites with ICCs of 0.98–1.00 Table . The maxSimilarly, in longitudinal analysis, compared to the cartilage volume change using 1.5 mm thick slices, decreasing the number of the slices by extracting one in two to one in four slices led to very little over or under estimation of the mean changes in cartilage volume at lateral, medial tibial and patellar sites Table . The meaThis study suggests that lateral, medial tibial and patellar cartilage volumes measured from up to one in four 1.5 mm thick slices are quite comparable to those obtained from 1.5 mm thick slices. If the agreement is defined at high levels expected to lead to minimal measurement error, then knee cartilage volume can be measured sufficiently and accurately with one in two slices both cross-sectionally and longitudinally regardless of ROA status and./or reader. This approach will lead to a substantial decrease in post-scan processing time and make large-scale studies of knee cartilage volume more feasible.Currently, there is no reported information on the number of the slices of MRI scans to measure cartilage volume apart from a recent paper from our own group which had similar findings to this study with different readers and geographic location . In a stIdeally, the more slices used, the more accurate the estimation of the object's volume, as they may contain more information. However, for a completely regular structure, such as a cylinder, the area of a single slice with length gives an exact volume. It is therefore reassuring but not surprising that the current study demonstrates a minimum reduction in the knee cartilage volume and volume change over time as tibial and patellar cartilages have a relatively regular structure. A different interpretation may apply to femoral cartilage and we do not have data on this imaging site.The current study simply examined the effect of decreasing the number of slices on the estimation of knee cartilage volume and volume change while all other variables were kept constant. We did not re-scan the study subjects but simply estimated the cartilage volume by using one in two, one in three, or one in four slices. This has an advantage of allowing us to examine the single effect of sampling intensity in the situation where all other variables such as re-positioning the subject and measurement were kept constant. The effect of these errors on measurement have been well-documented ,31. For The current study has a number of potential limitations. Firstly, which sampling intensity should be used in the MRI scan of knee cartilage depends on the purpose of the measurement. Our results cannot be applied to individual cartilage volume, but only for mean cartilage volume in groups as the individual difference in cartilage volume increases with decreasing sampling intensity. Secondly, decreasing sampling intensity will increase measurement error as the remaining slices focus on different portions of the irregularly shaped cartilage. Depending on what particular surfaces remain, however, the overall volume may be increased or decreased. If this is random, then the mean will remain the same as demonstrated in the current study. Thirdly, the ICC can be influenced by traits in the sample in which it is assessed. Age, sex and BMI have been reported to be associated with knee cartilage volume . These mKnee cartilage volume and its rate of change can be accurately measured with every second 1.5 mm thick MR slice. This approach will lead to a substantial decrease in post-scan processing time and make large-scale studies of knee cartilage volume more feasible.The author(s) declare that they have no competing interests.GZ designed and carried out the study planning, data collection, analysis and interpretation of the analysis, and preparation of the manuscript. CD participated in data collection and critical revision of the manuscript. FC participated in the study planning and critical revision of the manuscript. GJ designed the study, participated in analysis and interpretation of the analysis, and critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


SLC11A1 is involved in susceptibility to both autoimmune and infectious disorders. The aim of this study was to evaluate whether SLC11A1 has a role in the susceptibility to inflammatory bowel disease (IBD) by characterizing a promoter polymorphism within the gene and two short tandem repeat (STR) markers in genetic proximity to SLC11A1.Significant evidence suggests that a promoter polymorphism withinthe gene SLC11A1 genotype-phenotype correlations. Polymorphic markers were amplified from germline DNA and typed using gel electrophoresis. Genotype-phenotype correlations were defined using case-control, haplotype, and family-based association studies.The studied population consisted of 484 Caucasians with IBD, 144 population controls, and 348 non-IBD-affected first-degree relatives of IBD patients. IBD subjects were re-categorized at the sub-disease phenotypic level to characterize possible SLC11A1 disease association; most significantly, there was no apparent evidence of SLC11A1 promoter allele association in the studied Crohn's disease population.This study did not provide compelling evidence for SLC11A1 promoter polymorphisms are involved in susceptibility to this form of IBD.Our results therefore refute previous studies that have shown  Inflammatory bowel disease (IBD) encompasses a number of disorders that are characterized by chronic inflammation of the gastrointestinal tract. The three specific forms of the disease known as Crohn disease (CD), ulcerative colitis (UC), or the less well-characterized indeterminate colitis (IC), are diagnosed based on the results of clinical observations, histology, radiology, and to a lesser extent, serology. Prominent clinical features include profuse diarrhea, abdominal pain, and increased colorectal cancer risk. IBD affects an estimated one million Americans , with UCSLC11A1 in the susceptibility to IBD. SLC11A1 is a proton-coupled bivalent metal antiporter that is crucial in early macrophage activation [SLC11A1 encodes a highly hydrophobic 550 amino acid membrane protein and is located on the long arm of chromosome 2 (2q35), a region that has not been identified as being within an IBD susceptibility locus. Genetic studies have shown that different alleles of a (GT)n dinucleotide repeat polymorphism located within the promoter region of SLC11A1 cause susceptibility to different human diseases. Allele 2 of this polymorphism is associated with susceptibility to intracellular pathogen infection [The aim of this study was to investigate the role of the gene tivation . SLC11A1nfection , and allsome 2 2q5, a regiSLC11A1 are associated with CD [n promoter polymorphism, however, was not examined in that study. A recent study by Kojima et al [SLC11A1 has been implicated in susceptibility to IBD. Previous work from this laboratory  has show with CD . The escribed . These mThis prospective study was approved by the University of Louisville Institutional Review Board. Written informed consent was obtained from all subjects. Patients were derived from a University-based colon and rectal surgery practice. Initial IBD diagnoses were determined through radiological, endoscopic, and/or histopathological studies. Histology was available in all cases.The study population consisted of a total of 628 Caucasians, including 254 unrelated individuals with CD (63% women), 165 with UC (53% women), 65 with IC 70% women), and 144 population controls (74% women). Demographic data for the IBD population is shown in Table 0% women,A total of 348 non-affected first-degree relatives of IBD patients were available for the study. There were 63 CD families , 43 UC families , and 14 IC families . In addition, there were 17 CD families, 18 UC families, and 9 IC families who could not be classified as either a triad or discordant sibling pairs.SLC11A1 promoter polymorphism was performed using 100 ng template DNA, 2.5 mM MgCl2, 50 mM KCl, 10 mM of tris/HCl, 0.2 μM of each dNTP, 200 pmol of each primer, and 1 unit of Taq polymerase . Primer sequences were as follows: 5'GACATGAAGACTCGCATTAG3' & 5'TCAAGTCTCCACCAGCCTAGT3'. The product was amplified using the following conditions: 94°C for 10 min, followed by 25 cycles of 94°C for 30 sec, 55°C for 75 sec, and 72°C for 20 sec. A final extension step was run at 72°C for 6 min. Genotyping was performed using gel electrophoresis . PCR amplification of the STR markers D2S434 and D2S1323 was performed as described previously [STRs were amplified from genomic DNA extracted from peripheral leukocytes. Polymerase chain reaction (PCR) primers were custom-synthesized . PCR amplification of the o = 1, where πo is the proportion of tests in which the null hypothesis is true.Case-control analyses were performed using STR genotype-allele frequencies in cohorts subdivided on the variables outlined in Table only performed in those disease groups possessing a global test q-value <0.05. Therefore, 2 × 2 contingency tables were only constructed for genotypes with a global test q-value meeting this condition. Disease versus control comparisons were performed using Fisher's exact test and corrected for multiple testing using the q-value method as described above.To limit the number of individual disease-control genotype-allele comparisons, further analyses were Since IBD affecting the colon is the primary focus of our research, the L2 colonic CD and L3 ileocolonic CD groups were combined, enabling maximization of statistical power. We argue that this approach will not impair the validity of statistical analyses given that the Vienna Classification is somewhat arbitrary and that group genetic homogeneity may be increased by combining all those cases with colonic IBD. Furthermore, owing to the relatively low numbers of individuals with CD proximal to the terminal ileum , this disease subgroup was excluded from case-control analyses.Family-based tests used included the pedigree disequilibrium test (PDT) ,12 and aSLC11A1 promoter polymorphism STR D2S434 and STR D2S1323 were named numerically according to molecular weight, with "1" representing a larger allele than "2" and so on. Allele distributions are shown in Table Alleles for the SLC11A1 promoter polymorphism alleles identified in the study population revealed the following sequences:DNA sequence analysis of the three 5AC(GT)5AC(GT)11GGCAGA(G)6Allele 1: T(GT)5AC(GT)5AC(GT)10GGCAGA(G)6Allele 2: T(GT)5AC(GT)5AC(GT)9GGCAGA(G)6Allele 3: T(GT)These sequences corresponded to alleles 1, 2, and 3 as described by Searle and Blackwell . Allelesversus control comparisons of the studied markers are shown in Table n promoter polymorphism did not reveal any evidence of association. Additionally, we studied STR markers in genetic proximity to SLC11A1. The STR D2S1323 exhibited evidence of association with the test of the global null hypothesis of homogeneity of all groups exhibiting statistical significance for allele frequencies . To limit multiple testing, individual pair-wise disease versus control comparisons were only performed for D2S1323 alleles. These analyses showed over-representation of allele 1 in the UC group . The STR D2S434 did not reveal any evidence of association. Further analyses of genotype and allele data for all markers did not show a correlation with either age of onset or disease behavior.Results from global disease Family-based tests were performed for each of the three markers. Neither the likelihood ratio test nor the PDT revealed significant evidence of association for any of these markers.SLC11A1 promoter polymorphism alleles and any IBD sub-phenotypic group in this population, which unexpectedly refutes the significant evidence of others [SLC11A1 promoter polymorphisms to be involved in susceptibility to autoimmune disorders and disorders that are characterized by a high degree of immunological dysregulation, including IBD [SLC11A1 expression driven by alleles 1, 2, and 4; however, allele 3-driven SLC11A1 expression was enhanced. It has long been recognized that altered expression of SLC11A1 in response to LPS is a key event in the activation of macrophages upon contact with a variety of pathogens [No association was observed between any f others . This reding IBD . Searle ding IBD  investigathogens .SLC11A1 as an IBD candidate gene.Based on those observations, we hypothesized that over-representation of promoter allele 3 in CD patients could lead to hyperactivation of bowel wall macrophages that are chronically exposed to high levels of LPS. This could subsequently cause the autoimmune-like phenotype characteristic of IBD. This hypothesis must be rejected in this cohort due to the absence of association with promoter allele 3 in any IBD subgroup. Analysis of intragenic single nucleotide polymorphisms are required to conclusively exclude SLC11A1. This association was not, however, confirmed upon family-based statistical analysis. The current finding was particularly surprising to us, given that our previous study showed this STR to be associated with CD rather than UC [vice versa) may have been a confounding variable that may have in part led to what we now consider the false-positive association observed in the CD population. It should also be noted that the current study contains over two-fold the number of subjects as compared with the previous work, and thus statistical power has been significantly increased. Furthermore, our current statistical methodology is now far superior, and we argue that our stringent means of correcting for the effects of multiple testing effectively minimizes the likelihood of detecting a false-positive association. This is a vast improvement over our earlier study in which compounding of a type 1 error was not taken into account. Finally, genotyping in the earlier study was performed on specimens with a rather heterogeneous nature and consisted of genetic data derived from DNA extracted from both surgical specimens  and leukocytes . In our current study, genetic material has only been isolated from leukocytes, thus reducing the possibility of detecting variations within somatic DNA.D2S1323 was associated with UC in this population. Indeed, this was the only evidence for association in any of the studied STR markers in proximity to  than UC . The sigSLC11A1 (GT)n promoter polymorphism in IBD sub-phenotypic groups and accept the null hypothesis as confirmed by family-based testing. We did, however, identify an association between an allele of the STR D2S1323 and UC upon case-control analysis. This association was not confirmed on family-based and haplotype testing, and its actual significance in IBD remains to be seen.In summary, we have characterized the The author(s) declare that they have no competing interests.NPSC performed genotyping, PDT analyses, and drafted the manuscript; MRE performed genotyping and was responsible for coordination of laboratory work; authors DWC and RKL performed genotyping; GAC performed statistical analyses; REP performed re-review of colonic IBD histology; SG collected all patient samples, participated in study design, coordination, and manuscript preparation and obtained funding for this work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: