Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5′ ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3′ intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3′ ends of genes. Thus, recent intron dynamics do not support a model whereby 5′ intron positional bias is generated solely by 3′-biased intron loss. A comparative study of four fungal genomes reveals the patterns of intron gain and loss over several hundred million years of evolution Saccharomyces cerevisiae. It has been suggested that both the paucity and positional bias of introns in yeast may be due to intron loss through a mechanism of homologous recombination of spliced messages reverse-transcribed from the 3′ poly-adenylated tail , to roughly seven introns per gene on average for some basidiomycetes (e.g., Cryptococcus). Finally, fungi display a strong 5′ bias in intron positions, enabling us to investigate the processes underlying this phenomenon.Fungal genomes are in many ways ideal for exploring questions of intron evolution. The fundamental aspects of intron biology are shared between fungi and other eukaryotes, making fungi appropriate model organisms for intron study. They are gene dense with relatively simple gene structures compared with plants and animals, making gene prediction more accurate. Fungi also display a wide diversity of gene structures, ranging from far less than one intron per gene for SRY. Evidence for the occurrence of multiple independent intron losses has also been reported in studies such as those by Caenorhabditis elegans.In principle, a 5′ intron bias could arise through various combinations of intron gain and loss, and a complete understanding of intron positional bias requires an assessment of the contributions of both of these processes. A number of studies demonstrate the occurrence of intron gain and loss in individual genes or gene families. Fugu as an outgroup) and between mouse and rat (with human as an outgroup), and observed a sparseness of intron loss and complete absence of intron gain in these closely related organisms. On the other hand, More recently, a number of genome-wide studies of intron dynamics have been conducted. Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, and Neurospora crassa. Ortholog sets composed of one gene from each of the four genomes were identified as pairwise best bidirectional BLAST hits satisfying stringent overlap criteria. Orthologs in each set were subsequently aligned, and the locations of introns were marked. These intron positions  were subjected to rigorous alignment quality filtering to eliminate alignment and annotation errors  passed the quality filter. The complete set of aligned orthologs with passing and failing intron positions is provided in N. crassa (blue box), and the third is present only in A. nidulans, and, because of the ambiguity in inferring gain or loss in this case, is classified as “Other” (black-edged gray rectangle). Examining the region around the one raw gained intron in N. crassa at the nucleotide level  and M. grisea (M) but absent from F. graminearum (F) and N. crassa (N).where r is defined for a given organism as the fraction of introns in the parent that did not survive in any sibling. We define “sibling raw losses” for an organism as the number of introns that are present in the organism and at least one cousin but in no sibling. This quantity is then divided by the number of introns present in that organism and at least one cousin to give the sibling loss rate. For example, the sibling loss rate for F. graminearum is given byThe sibling loss rate, m, and subtract this quantity from the raw gains to obtain “inferred gains.” To calculate m we first count B0, the number of introns conserved in the organism and at least one sibling, but in no cousin. The quantities m and B0 are related through the variablen0, the number of introns present in an organism's parent but not in any cousin, by the equationsWe next correct the raw gains for each organism. Raw gains include some introns that were in fact lost in all but one lineage. We use the loss rates to calculate the expected number of these multiple losses, andThis follows from our assumption of independent gains and losses. Thus, we can calculate the expected number of multiple losses asn1, and the number not present in any cousin n0 (introduced above). To estimate the size of the set of parental introns present in at least one cousin, we first count the subset of these introns that are presently observable. An intron is in this set if it is present in at least one cousin and at least one sibling, or is present in at least one cousin and in the organism in question. We call this number of introns B1. By the assumption that gains and losses are independent, we haveWe use the loss rates to estimate the number of introns in each organism's parent. To do so, we estimate separately the number of parental introns present in at least one cousin Using this relation and the one in F. graminearum as an example, the true number of losses would also include some introns conserved in the patterns A, M, N, and MN. We calculate the number of inferred losses as ntotal q.Finally, we correct raw losses. Our definition of raw losses undercounts the true number by omitting those introns not conserved in at least one cousin and at least one sibling. Taking This method can be extended to any phylogenetic tree and to any organism with at least one cousin.M. grisea  than the average for other genes analyzed, resulting in unusually large numbers of introns in these genes  synthetase genes. These genes encode a widely conserved protein that catalyzes the production of PRPP, a precursor in the nucleotide biosynthesis pathway. In contrast to the majority of orthologs that displayed fewer than two gained introns, the set of PRPP synthetase genes displayed a total of 22 raw gains (se genes B. In com(M. grisea,N. crassa, F. graminearum), we determined the frequency of phase 0, 1, and 2 introns in the set of all intron positions  based on parsimony. However, this does not accurately account for the possibility of multiple gain or loss events. We have developed a probabilistic model that allows for multiple events, providing a corrected estimate of the total number of gains and losses within the dataset. Our probabilistic method allows for a more accurate assessment of rates of gain and loss. In our dataset, allowing for multiple events results in only modest corrections to the rates estimated using parsimony.F. graminearum and M. grisea the number of introns gained was on par with the number lost and similar in magnitude to the number of introns gained in N. crassa.Our analysis demonstrates a significant role for intron gain over the past few hundred million years in the fungi analyzed. Previous analyses of specific gene families have provided evidence of specific instances of gained introns . HoweverThe mechanisms underlying intron gain are not known. We analyzed the set of predicted intron gains for possible signatures that might shed light on this process. No statistically significant bias was detected in the positions of gained introns along the coding sequence see , and no N. crassa and M. grisea suggests the possibility of independent increases in gain rate in the two species. Alternatively, the apparent high intron gain rate exhibited by this gene may have arisen just prior to the last common ancestor of N. crassa and M. grisea. Although it is premature to speculate about possible mechanisms, one possibility is that a factor or factors responsible for intron insertion evolved to associate with the PRPP synthetase gene locus, transcript, or message at this point, leading to a higher rate of intron insertion in this gene.Our data further indicate that intron gain can vary substantially between different gene families in a lineage-specific fashion. The PRPP synthetase gene is a particularly striking example, exhibiting significant increases in gained introns in two of the four lineages investigated. Moreover, the paucity of intron positions shared between Finally, our results do not support the mechanism commonly proposed to account for the 5′ positional bias of introns in intron-poor organisms . Contrarhttp://www.broad.mit.edu/annotation/fungi/fgi). The following datasets were used: A. nidulans (Assembly 1, 18 February 2003), N. crassa (Assembly 3, 1 February 2001), F. graminearum (Assembly 1, 11 March 2003), and M. grisea (Assembly 2, 18 July 2002).All sequences and annotations were taken from the Broad Institute Fungal Genome Initiative website . We repeated our analysis, requiring that each best bidirectional hit also be the only BLAST hit in each genome (spanning 60% the length of the longest protein). This protocol yielded only 1,178 ortholog sets, but gave qualitatively similar results for intron gains and losses .The proteins in each ortholog set were aligned using ClustalW 1.82 , and intRegions of low alignment quality were eliminated with a filter that required at least 30% identity and 50% similarity in a window of ten residues on each side of the intron position. These parameters were determined following manual classification of a set of 181 randomly selected intron positions as “clearly homologous,” “ambiguous/possibly homologous,” or “non-homologous” see B. Using To further exclude likely annotation and alignment errors, intron positions were also filtered by eliminating positions adjacent to gaps in the amino acid alignment and by eliminating positions with nearby introns but low evidence of homology in the intervening sequence. It is possible that some of these positions may in fact reflect intron gain or loss events that occurred simultaneously with coding sequence insertion or deletion. However, removing these positions did not significantly impact our results, as the number of positions adjacent to gaps was only about one-tenth of the number of positions that passed the quality filter, and the introns removed did not have an apparent positional bias see A( p < 3 × 10−22 for M. grisea,p < 4 × 10−9 for N. crassa, and p < 0.007 for F. graminearum). In the second case λ is given by the total number of raw gains observed in that organism multiplied by the length of that gene in amino acids and divided by the total number of amino acids in all genes in that organism ( p < 7 × 10−25 for M. grisea,p < 3 × 10−10 for N. crassa, and p < 0.003 for F. graminearum). We reported the less significant of the two p-values in the results.We modeled the number of gains in a particular organism as a Poisson distribution under two different null hypotheses. One null hypothesis was that the gains were spread uniformly across all genes. The other was that the number of gains in each gene was proportional to the length of the gene. In the first case the Poisson parameter λ is given by the total number of raw gains observed in that organism divided by the total number of ortholog sets p-value for the significance of phase 0 bias was determined by the χ2 test with two degrees of freedom using equal expected phase frequencies. To detect sequence bias at intron insertion sites, we examined gained introns separately in F. graminearum,M. grisea, and N. crassa. For each gained intron, we extracted four bases upstream and downstream of orthologous sites in the other two sequences, consistent with For each of the three in-group lineages, the frequency of phase 0, 1, and 2 introns was determined for five different datasets: for each class of conservation (conserved, raw gains, and raw losses), for all introns passing our filter, and for all introns in the ortholog set. The Figure S1Positional biases in intron gain, loss, and current distribution in three fungal genomes determined using orthologs predicted by a “bidirectional only hit” method. (A), (B), and (C) are roughly analogous to (D), (E), and (A), respectively, in (78 KB DOC).Click here for additional data file.Table S1http://genes.mit.edu/NielsenEtAl/.Also available at (4.3 MB ZIP).Click here for additional data file.


To evaluate the clinical outcome of primary endonasal laser assisted dacryocystorhinostomy (ENL-DCR) using the potassium-titanyl-phosphate laser.We retrospectively reviewed all primary ENL-DCRs performed within a period of twelve months by the same combined Ophthalmology and Otorhinolaringology team in Freeman Hospital, Newcastle upon Tyne, UK. The main outcome measure for success was resolution or significant improvement of epiphora. Details of surgery, intraoperative and postoperative complications, as well as pathology associated with failure were also studied. Patients were followed up for at least 12 months.A total of 41 consecutive ENL-DCRs on 29 patients  were analysed. All patients had bicanalicular silicone intubation for at least 4 months. The success rate at 12 months postoperatively was 78.1%. Pathology associated with failure included: intranasal pathology (12.2%), mucocele (7.3%), and systemic sarcoidosis (2.4%). No significant intra-operative complications were recorded.The ENL-DCR with potassium-titanyl-phosphate laser can be considered as a safe and efficient primary procedure for the treatment of nasolacrimal duct obstruction. External dacryocystorhinostomy (EXT-DCR) was first described by Toti ,2 in 190emostasis.Reported primary ENL-DCR success rates vary from 68% to 99% ,6,11, deThe records of all 47 ENL-DCR procedures that were performed in Freeman Hospital, Newcastle upon Tyne, United Kingdom, within a period of twelve months were retrospectively studied. The patients' main symptom was moderate to severe epiphora. Obstruction of the nasolacrimal system distal to the lacrimal sac was diagnosed with nasolacrimal syringing. Radiographic imaging was not part of the routine preoperative evaluation. Otorhinolaryngological preoperative assessment included full endoscopic examination of nasal cavities, looking for evidence of mucosal disease including polyps particularly in the middle meati. Exclusion criteria for ENL-DCR were: noticeable lower lid laxity, previous lacrimal surgery, suspicion of malignancy and previous radiation therapy. Each patient with primary nasolacrimal duct obstruction was counselled as to the advantages and disadvantages of EXT- DCR versus ENL-DCR, together with estimated success rates of the two different types of DCR. The operations were performed by the same ophthalmologist (C.N.) and ENT surgeon (S.C.). The majority of patients had surgery under local anaesthesia on an outpatient basis, except for one who opted for general anaesthesia. For local anaesthesia, Amethocaine drops were instilled in the conjunctival sac, followed by injection of Xylocaine 2% with 1:200000 Adrenaline in the medial third of both eyelids and transcaruncularly to the lacrimal sac. Cophenylcaine spray and intranasal cocaine 4% paste was applied to achieve anaesthesia and haemostasis. Dilatation of the lower punctum was performed and a 20G vitreoretinal probe was inserted in the lower canaliculus and advanced into the nasolacrimal sac. The light was directly visualised endonasally with a 0° rigid nasal endoscope, and the laser energy was delivered, with full laser precautions, via a KTP laser probe guided by the light. The nasal mucosa and lacrimal bone were ablated and the ostium was enlarged anteriorly as necessary with a microronguer. Bicanalicular O'Donoghue silicone tubes were inserted and secured with a Watzke sleeve. A course of topical Chloramphenicol drops was given for 1 week. Patients were examined 1 week postoperatively and then at 6 months for removal of tubes, or earlier, if discomfort was experienced. Mean follow up period was 16 months (range 12–24 months).Forty- seven consecutive primary ENL-DCR operations with lacrimal intubation were performed from March 2001 to February 2002 on 35 patients with primary acquired nasolacrimal duct obstruction. Five cases with incomplete follow up and one which had to be converted to EXT-DCR due to very thick lacrimal bone were excluded from the study. Forty-one ENL-DCR procedures on 29 patients  were included in the study. Mean age was 75 years (range 47–90, SD 13.7). 12 patients (41.4%) underwent simultaneous bilateral surgery and 17 (58.6%) had unilateral ENL-DCR. In total, 19 right sided (46.3%) and 22 left sided (53.7%) procedures were recorded  and previous dacryocystitis in 4 (9.8%), while in another 3 cases (7.3%) these two conditions co-existed Table . Mild meDuring the operation the mean laser energy used was 400.2 joules (range 96–797, SD 188.5). Serious intra-operative complications did not occur, although in some patients' records per-operative mild discomfort was documented.The removal of silicone tubes was scheduled at 6 months post-operatively, though in ten patients extubation was performed at 1–4 months due to discomfort. In two patients, mild nasal haemorrhage was noted on removal of tubes.The operation was defined as being successful if the patient was asymptomatic or if there was significant improvement of symptoms, not requiring any additional procedure. Success rate at 12 months postoperatively was 78.1%. No improvement of symptoms was noted in 8 patients (19.5%), while 1 patient reported worsening of epiphora Table . PatholoENL-DCR is a well established surgical technique with some advantages compared to the conventional EXT-DCR. These include limitation of tissue injury to the discrete fistula site, avoidance of a skin incision, excellent haemostasis, the ability to perform a lacrimal bypass operation on an outpatient basis, quicker patient rehabilitation, decreased overall health care expense and patients' preference ,13. MainENL-DCR avoiding the use of laser is a well described procedure. Several surgical instruments have been employed to remove the bone overlying the lacrimal sac, including drills, osteotomes, curettes and rongeurs. Weidenbecher reported resolution or improvement of symptoms in 95% of the patients. In anot2, Ho:Yag, Nd:Yag), have been employed in an attempt to achieve better bone ablation and haemostasis. Massaro et al [Various types of laser , where iThe optimum duration of stent retention following DCR is controversial. In published series it varies from 4 weeks to 6 months, though there is some evidence that prolonged silicone intubation may increase the incidence of DCR failure by inciting a granulomatous reaction at the internal ostium, with subsequent stenosis . All ourReported pre-operative risk factors for ENL-DCR failure include pre-existing sinus disease, mucocele, nasal septum deviation, connective tissue diseases such as sarcoidosis, previous EXT-DCR, other nasal surgery, nasal fracture, and thickened lacrimal bone . In our It has been proposed that the thermal energy produced by the laser may lead to scarring and subsequent blockage of the ostium . It has ENL-DCR performed under local anaesthesia is reported to be generally well tolerated by the patients. In our In our experience, the use of KTP laser in ENL-DCR under local anaesthesia with the adjunctive use of an osteotome is a safe and efficient technique, with good results. The particular advantages of this laser are its superior haemostatic properties and its diversity, which reduces the cost of the operation, as it is already employed in other procedures by the ENT surgeons. Significant complications are not common with this technique. In cases of failure, revision ENL-DCR or EXT- DCR can be performed. ENL-DCR with KTP laser is routinely performed under local anaesthesia, thus avoiding the risks of general anaesthesia usually required for EXT-DCR  The operENL-DCR using KTP laser appears to be an efficient technique, with low complication rate and it is well tolerated by the patients. It still needs refinement in order to achieve the higher success rate of the EXT- DCR, which remains the gold standard method for the treatment of nasolacrimal duct obstruction. At present, we believe that patients should be involved in the decision on the type of operation, after comprehensive consultation on the advantages and disadvantages of each technique.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression. A computational analysis of human RNA has identified 1,445 transcripts are edited mainly within non-coding Alu repeats, with the potential effect of regulating alternative splicing On the molecular level, the complexity of higher organisms is based on the number of different gene products available for structural, enzymatic, and regulatory functions. Posttranscriptional and/or posttranslational mechanisms have an important role in generating RNA and protein diversity . One posCaenorhabditis elegans resulted in transgene silencing, suggesting that the RNA editing and RNA interference (RNAi) pathways intersect  discrepancies in these cDNAs are the result of RNA editing involving intramolecular pairs of inverted Alu repeat sequences, repetitive elements that represent approximately 10% of the human genome and are concentrated in and around genes .We also characterize functional consequences of the observed editing events and the factors that determine editing levels in Alu repeats and their modification patterns. The prevalence of Alu elements in primate genes, together with our experimental and computational analysis, suggests that the vast majority of primary human gene transcripts (greater than 85% of RNAs with average structure) are subject to A-to-I RNA editing. We show how editing might influence the alternative splicing of exonized Alu elements and discuss the implications of this extensive modification of mRNAs bearing repetitive elements for the regulation of gene expression.A hallmark of an A-to-I RNA editing event is an AtoG transition when comparing genomic and cDNA sequences of the affected gene since inosine base-pairs with cytosine and therefore is replaced by guanosine during reverse transcription and PCR amplification. However, AtoG discrepancies between genomic and cDNA sequences can also be due to single-nucleotide polymorphisms (SNPs) or errors in databases. Therefore the search for edited sequences on a genome-wide basis is not feasible solely based on this single feature. However, in some cases of editing, not a single, but a cluster of AtoG discrepancies between genomic and cDNA sequences is evident within a stretch of a few hundred nucleotides . TherefoIn an initial screen for candidate genes, we used the Human Unidentified Gene-Encoded (HUGE) database of ca. 3,000 human cDNAs derived from the Kazusa cDNA sequencing project . SeveralC. elegans  containing pairs of Alu repeats as seen schematically in Many primary gene transcripts allow several energetically favorable foldback structures to be predicted for a given Alu that involve different combinations of Alu pairs. Do all these alternative Alu-pair foldback structures exist in vivo and are therefore subject to RNA editing? To address this question we examined the editing pattern of the G-protein-coupled receptor 81 . GPR81 contains four Alu elements, one sense and three antisense oriented, in the 3.6-kb pre-mRNA and was selected based on Alu repeat configuration and transcript length. If the alternative foldback structures depicted in These results suggest that Alu elements in human mRNAs are subject to RNA editing by ADARs because of foldback structures formed between two oppositely oriented Alus present within the same primary transcript.Exonic Alu repeat elements are predominantly located in the 5′- and 3′-UTRs of mRNAs, and as a result, most cases of Alu editing occur in noncoding regions. However, some editing events predict amino acid changes . Among tThe LUSTR1 cDNA codes for a G-protein-coupled seven-transmembrane receptor , with three AtoG discrepancies located within an alternatively spliced AluJo-derived exon that leads to the in-frame insertion of 29 amino acids between transmembrane regions V and VI of the protein see A. The exAnalyzing the RNA editing pattern of LUSTR1 pre-mRNA revealed additional intronic editing sites, one of which represents the splice acceptor adenosine (AG to IG) in intron 15  is generated in 57% of primary transcripts by RNA editing. This is predicted to give rise to alternatively spliced mRNA products represented by GenBank entry AF385431 see B. This iIt is intriguing that we find cases where editing in alternatively spliced Alu exons, or within adjacent splice sites, interferes with or counteracts exon formation of an Alu repeat. It suggests that RNA editing might be more generally involved in the regulation of Alu exonization. Recently, it has been shown that more than 5% of the alternatively spliced exons in the human genome are Alu derived . ExonizaFurthermore, RNA editing in Alus might be involved also in the generation of novel introns as seems to be the case in GPR81. Statistically, however, the exonization of intronic Alus would be much more frequent than the intronization of exonic Alus because of the abundance of Alus in introns.The results presented above show that clusters of AtoG mismatches in cDNA/gDNA sequence comparisons represent an effective way to identify authentic editing cases with a low rate of false positives. Since all clusters of AtoG discrepancies mapped to repeat elements, we wondered how prevalent the editing of Alu or other repeat elements is in the human transcriptome. Therefore, we devised a database search procedure to identify pairs of inverted repetitive elements in human mRNAs exhibiting AtoG transitions.Initially, a limited search was carried out for closely spaced (less than 2 kb) inverted pairs of human Alu, MIR, and L1 repeat elements that overlap with exonic sequences and for which an mRNA sequence can be found in GenBank entries. This search, involving about one-third of all repeat elements in the human genome, identified 71 mRNAs with exonic repetitive-element pairs (51 Alu, six L1, six MER, and eight MIR). From those mRNAs, 27 displayed clusters of AtoG changes, all in Alu elements. Fourteen of these genes were chosen for experimental analysis, and all 14 proved to be subject to A-to-I RNA editing . Since tWe analyzed the total of 103,723 human mRNA sequences  are approximately three times more frequent than transversions is in line with results from previous studies analyzing gDNA sequences , there i2 test comparing the observed number of AtoG discrepancies with the expected number, based on the number of non-AtoG mismatches present in the same sequence. Elements with a χ2 higher than the critical value for α = 0.00001 (corresponding to a probability of one in 100,000 that the observed AtoG transitions are due to SNPs or sequencing errors) were selected as “edited” and will be called so throughout the rest of the manuscript. Using this approach we found that out of those 17,406 mRNAs with one or more exonic Alu elements, 1,445 (8.0%) mRNAs are edited within one or more of the Alu sequences  between cDNA and gDNA. The probability that a cluster of several AtoG discrepancies is due to sequencing errors or SNPs  is negligible. Thus the number of clustered AtoG changes can be used to distinguish genuinely edited elements from elements with aberrant or non-editing-related base changes. For each Alu element with AtoG discrepancies, we computed the χIn order to validate our screening approach, we performed an identical analysis for GtoA, CtoT, and TtoC mismatches. Compared to the 1,925 AtoG-edited Alu elements in mRNA, we found 12 GtoA, 11 CtoT, and 11 TtoC cases of “editing.” These cases may represent false positives and thus set the error level of our screen to less than 0.6%.These results suggest that out of the 103,723 human mRNAs at least 1.4% are A-to-I edited within an exonic Alu element. Apart from Alu repeats, many more low- and high-frequency repeats exist in the human genome  and migh19.3 or 94.7% (85.0% with the 12.4 Alu/gene estimate). While this value is an approximation, assuming that all genes have similar structures, and does not take into account editing in other repeat elements, it does reflect the magnitude of repetitive-element editing.Most Alu repeats are located in introns, and it is there where the bulk of RNA editing is expected to occur. The average number of Alu repeats per gene is 12.4 estimated for Chromosomes 21 and 22 . This vaTo gain insight into the factors that determine which Alus are subject to RNA editing, and under what circumstances, the identified set of 1,925 high-confidence cases of editing in Alu elements  or its orientation in relation to the mRNA (sense, antisense). No significant correlation of Alu editing was detected with any of these features (data not shown).The availability of such a large collection of A-to-I edited sequences resulting from this analysis allowed us to examine the modification pattern of edited Alu elements for potential editing hot spots or base preferences. To this end we first aligned all 141 edited Alu sequences (greater than 260 bp) in RNAs originating from Chromosome 1 and mapped the edited sites on their consensus sequence . InteresAs a result of the high CpG mutation rate, frequently the Alu foldback structure of the unedited RNA is predicted to carry A–C mismatches at these positions. Editing at these sites restores the CpG repeat (CA→CI) on the RNA level and converts the A–C mismatch to an I–C base pair. Energy calculations for several predicted Alu pairs show, surprisingly, that the stability of the foldback structure is not diminished by editing but often increased because of the high frequency of I/C pair formation (data not shown). It is therefore unlikely that in the case of Alu foldback structures, RNA editing serves to resolve RNA secondary structures that interfere with the processes of splicing or translation of these RNAs, as suggested previously . Two typcis preferences was carried out by extracting 14,774 pentanucleotide sequences with the edited adenosine as the middle base and estimating the frequency of each base at positions −2, −1, 1, and 2 relative to the editing site. To correct for Alu sequence bias we performed the same analysis for a randomly chosen adenosine for each edited adenosine in our sample. We then subtracted those frequencies to obtain unbiased editing preferences  are very low compared to editing levels seen in Alu repeats (see For mRNA fractions, we estimated the inosine content due to Alu editing as follows: In 103,724 mRNAs we found 23,204 AtoG mismatches, while the same sequence sample has an average for the other transitions of 3,271. Assuming an average mRNA size of 4 kb, the ratio of inosine in the sample is estimated to be one inosine every 20,814 nucleotides (103,724 × 4,000/[23,204–3271]) generated by editing in Alu sequences. This estimation for Alu editing is in the range of one inosine in 17,000 nt (brain), one in 33,000 nt (lung, heart), to one inosine in 150,000 nt  as was experimentally determined by Bass and colleagues in the polyA-fraction of rat RNAs . Since tWhile a significant amount of editing occurs in mRNAs that contain repetitive elements in their exons, our results predict that the bulk of A-to-I editing takes place in intronic sequences missing from cDNA databases. This is suggested by the experimental results regarding the LUSTR, GPR81, p53, SIRT2, NFκB, and paraplegin genes, for which intronic data was available see , and 5A.It has been shown that hyperedited, inosine-containing RNAs are retained in the nucleus by a protein complex containing the inosine binding protein p54 . In viewC. elegans where inactivation of the editing machinery leads to transgene silencing . Total RNA isolation and reverse transcription have been described previously . Gene-spFor analysis of the pool of human cDNA sequences we developed a program named Procedures for Repetitive Element Foldback Analysis (PREFA). We used the set of cDNA sequences from the UCSC database (July 2003) comprising 103,723 sequences . The set of repetitive elements (for Alus 1,163,041 unique elements) and related information of the human genome  were discarded as misaligned or erroneous. From the repetitive elements showing at least a single AtoG change we selected those where mismatch distribution cannot be accounted for by SNPs and sequence errors using the following procedure:The overall expected ratio of AtoG discrepancies relative to the total number of mismatches was calculated from the whole sample, assuming the expected AtoG mismatches to be approximately equal to the average of the rest of the transitions:The expected probability for an AtoG mismatch at a single position in a given element was calculated from the total number of mismatches found in the element in cases where other mismatches were present (2) or from the whole sample where only AtoG mismatches were found (3): Here nAtoG and nOther is the total number of AtoG and non-AtoG mismatches found for this element:p for an AtoG mismatch to occur at any given position, the expected values for the number of AtoG were calculated:Given the probability 2 test was calculated for each element and those with a χ2 value exceeding the critical value (for α = 0.000001) were selected as edited, and these values correspond to approximately more than five AtoG changes in the absence of any other change in the approximately 300 bp of an Alu).A χFor each element in the high-confidence set the closest inverted element was identified among the elements present in the same gene boundaries. The distance separating the pair was calculated from the location of the first base of each element, according to the genomic sequence numbering and irrespective of their orientation. The divergence of each element was derived from the corresponding entry in the UCSC annotation database (ChrN_rmsk) representing mismatches per hundred bases. Tissue of origin of the RNAs was also derived from the UCSC mRNA annotation. For RNAs described to originate from multiple tissues, the corresponding RNAs were included in the count for each of those tissues. RNAs originating from a specific subregion of a tissue, such as subareas of the brain, were counted within the subregion but not in the whole-tissue set of RNAs.Alignment of the Chromosome 1-derived Alu sequences was performed with the MegAlign program of the DNASTAR (Madison, Wisconsin, United States) package (Lasergene) using the CLUSTAL algorithm . FurtherTable S1http://genome.ucsc.edu/cgi-bin/hgGateway and following the link to mRNA/Genomic alignment. We found that six cDNAs map on two chromosomes (AB095924, AK021666, AK055562, AK092837, AK094425, and BC039501); details are given for the most plausible assignment. We have observed that in the 43 cases that we experimentally analyzed, usually additional editing sites were identified when directly sequencing gene-specific PCR products.The database lists the GenBank accession numbers, gene names, gene product description, chromosome location, and type of Alu element and location within the mRNA sequence, the identity of the most likely pairing Alu elements within the same gene, and the distance in base pairs (bp) between the pairing Alus. The positions of all predicted editing sites within the individual sequences can be viewed by pasting the accession number into the USCS genome browser  at http:(276 KB XLS).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genetic sequences discussed in this paper are LUSTR (AB046844), KIAA0500 (AB007969), BTKI (AB037838), KIAA1497 (AB040930), and GPR81 (BC0067484).The GenBank ((http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) ID numbers for ADAR1, p53, SIRT2, NFκB, and SPG7 are 103, 7157, 22933, 4790, and 6687, respectively.The Entrez Gene (


WRKY proteins are newly identified transcription factors involved in many plant processes including plant responses to biotic and abiotic stresses. To date, genes encoding WRKY proteins have been identified only from plants. Comprehensive search for WRKY genes in non-plant organisms and phylogenetic analysis would provide invaluable information about the origin and expansion of the WRKY family.Giardia lamblia, a primitive eukaryote, Dictyostelium discoideum, a slime mold closely related to the lineage of animals and fungi, and the green alga Chlamydomonas reinhardtii, an early branching of plants. This ancestral WRKY gene seems to have duplicated many times during the evolution of plants, resulting in a large family in evolutionarily advanced flowering plants. In rice, the WRKY gene family consists of over 100 members. Analyses suggest that the C-terminal domain of the two-WRKY-domain encoding gene appears to be the ancestor of the single-WRKY-domain encoding genes, and that the WRKY domains may be phylogenetically classified into five groups. We propose a model to explain the WRKY family's origin in eukaryotes and expansion in plants.We searched all publicly available sequence data for WRKY genes. A single copy of the WRKY gene encoding two WRKY domains was identified from WRKY genes seem to have originated in early eukaryotes and greatly expanded in plants. The elucidation of the evolution and duplicative expansion of the WRKY genes should provide valuable information on their functions. Transcriptional control is a major mechanism whereby a cell or organism regulates its gene expression. Sequence-specific DNA-binding transcription regulators, one class of transcription factors , play anIpomoea batatas) [Arabidopsis thaliana [Avena fatua) [Dactylis glomerata) [Hordeum vulgare) [Nicotiana tabacum) [Matricaria chamomilla) [Oryza sativa) [Petroselinum crispum) [Retama raetam) [Saccharum hybrid cultivar) [Solanum dulcamara) [Solanum tuberosum) [Triticum aestivum) [Caenorhabditis elegans and Drosophila melanogaster) and yeast (Saccharomyces cerevisiae) [WRKY proteins are recently identified transcriptional regulators comprising a large gene family . The firbatatas) . Numerouthaliana ,6, wild a fatua) , orchardomerata) , barley vulgare) , tobaccotabacum) -13, chammomilla) , rice (O sativa) ,15, parscrispum) ,17, a de raetam) , sugarcaultivar) , bitterslcamara) , potato berosum) ,22, and estivum) . In addiestivum) . To datecis element, as a transcription activator [The WRKY family proteins contain one or two highly conserved WRKY domains characterized by the hallmark heptapeptide WRKYGQK and a zinc-finger structure distinct from other known zinc-finger motifs . To reguctivator .In plants, many WRKY proteins are involved in the defense against attack from pathogenic bacteria ,23,25-27Based on the number of WRKY domains and the pattern of the zinc-finger motif, Eulgem et al.  classifiGiardia lamblia, a primitive protozoan, and Dictyostelium discoideum, a slime mold. The data indicate an early origin of WRKY genes in eukaryota and tremendous gene amplifications in the plant lineage. We then cataloged the WRKY genes from the rice genome and compared them with Arabidopsis WRKY genes. We also identified WRKY genes from expressed sequence tags (ESTs) and EST-assembled sequence contigs from nineteen plant species. The result suggests that WRKY gene duplication events correlate with the increasing structural and functional complexities in land plants. We propose a model for the evolution of WRKY genes.Given the large family of WRKY genes with divergent regulatory functions in important plant processes, it would be desirable to understand the evolutionary origin and gene duplications leading to the multi-member WRKY family. The clarification of the phylogenetic relationships among WRKY genes in model plants will also assist understanding of the functions of these genes in important crops. We have comprehensively searched all currently available sequence data for the existence of WRKY genes outside the plant kingdom. Homologues of WRKY genes are found from two eukaryotic species: We searched for WRKY genes in two comprehensive datasets, GenBank's non-redundant (nr) and dbEST of all species. Together these datasets contain over 13 million sequence records from more than 110,000 organisms . HomologGiardia lamblia [Giardia is one of the most primitive organisms that represent the earliest branching among extant eukaryotes [Dictyostelium discoideum [D. discoideum belongs to the Mycetozoa, a lineage more closely related to animals and fungi than to green plants [Interestingly, two WRKY homologues were identified from non-plant eukaryotic species, and both have two WRKY domains [see Additional files  lamblia . The unikaryotes ,40. The scoideum . The genscoideum , indicatn plants ,43.Chlamydomonas reinhardtii is a unicellular green alga with a cell wall. It also has chloroplasts for photosynthesis. The evolutionary position of the species is located before the divergence of land plants [d plants ,45. The d plants . Since td plants  and 2. MRice, one of the most important crops for world agriculture, is recognized as a model monocot for the study of cereal crop genomes. A comprehensive catalog of rice WRKY genes would provide a basis for investigating the evolutionary patterns of the gene family and for transferring knowledge of the functions of these transcription factors from Arabidopsis to rice and from rice to other cereal crops.Giardia lamblia, and the conserved WRKY domain patterns can be found in A. thaliana [We identified the members of the WRKY family in rice (Japonica variety) from its published genome sequence . The WRKthaliana  as the rthaliana  . We then estimated the number of unique WRKY genes for 17 species using their Gene Indices, which are assembled EST sequence contigs with the minimal redundancy, provided by The Institute for Genomic Research (TIGR) . The anaable see . For theg plants .ESTs encoding WRKY proteins were identified in all the 19 species. Moreover, multiple WRKY genes are represented in the EST or contig sets for most plants including the moss and pine, with the most WRKY genes (109) from soybean see . AlthougThe abundance of WRKY ESTs in the total EST set is lower for pine, fern and moss than for flowering plants, as the percentage of WRKY ESTs in the total EST set for the three non-flowering plants is among the lowest see . The WRKThe identified WRKY genes were phylogenetically classified into five groups see . In six G. lamblia, the slime mold, the green alga, Arabidopsis and rice. The phylogenetic relationships were also inferred with the programs of the least squares and parsimony from PAUP 4.0 for the corresponding nucleotide sequences. We also did the same analysis for the rice dataset alone. The topology of trees obtained from these analyses is essentially the same, and the neighbor-joining tree is shown in Figure To examine the evolutionary relationships among the WRKY domains, we estimated the phylogeny by using the neighbor-joining program from PHYLIP 3.57 for the amino acid sequences of WRKY domains from In flowering plants, genes encoding WRKY domains appear to have been duplicated independently in monocots and dicots. For Group 3 domains, three subsets each of which consists of five or more members only from rice can be distinguished from the phylogram shown in Figure G. lamblia and the mycetozoan D. discoideum, but not in fungi and animals. G. lamblia is a primitive unicellular eukaryote diverged ~ 1,500 million years ago (mya) [WRKY genes seem to be an innovation in eukaryota after the divergence of eubacteria – archaea – eukaryota. In eukaryotes, the WRKY genes are present in the green plants as well as in the ancient eukaryote go (mya) . Originago (mya) ,3, the Wgo (mya) ,43, the G. lamblia, the slime mold and the green alga seems to be a Group 1 gene encoding two WRKY domains. The conservation of the C- and N-terminal domains suggests that they are derived from a single domain by domain duplication. Therefore we hypothesize that the earliest WRKY factor had one WRKY domain and the gene was innovated post the first appearance of eukaryotes ~ 2,500 mya [Giardia protist, ~ 1,500 mya. Second, our data and the previous results by Eulgem et al. [Based on the current data, we propose a model for the origin and evolution of the WRKY factor family Figure . First, ,500 mya  but priom et al.  suggest m et al. . Third, m et al.  and the m et al.  suggestsThe classification of the WRKY family in Arabidopsis by Eulgem et al.  is not cOur evolutionary analysis of WRKY transcription factors in this study may be important to the understanding of the overall mechanisms of biodiversity in the plant kingdom and the particular functions WRKY genes play in plant regulatory networks. First, the comparative analysis of WRKY factors in lower and higher plants indicates that the WRKY family expands as plants evolve from simpler, unicellular to more complex, multicellular forms. Since WRKY genes seem to play important regulatory roles in plants under abiotic and biotic stresses, and flowering plants which have the largest WRKY family are dominant over non-flowering plants in their distribution on the earth, WRKY genes might be essential for much of the enhanced adaptability of flowering plants to the environment. In comparison with pine, fern and moss, WRKY ESTs of flowering plants seem to be over-represented see , suggestOriginally believed to be plant-specific, WRKY transcription factor family has an early origin in eukaryotes and is also present in a slime mold which is more closely related to the lineage of fungi-animals than to plants. WRKY genes have been duplicated many times during evolution in plants, resulting in a large gene family for WRKY proteins in flowering plants. The elucidation of the evolutionary pathway of WRKY family and a new classification system we proposed based on phylogenetic analysis, conserved WRKY domain structures and intron positions should assist the functional characterization of WRKY genes.Oryza sativa spp. japonica) (OSA1, released on 7/27/2003) and Arabidopsis (ATH1, released on 4/17/2003) were downloaded from TIGR [Chlamydomonas reinhardtii, the genome sequence release 1.0 on 2/4/2003 was used [Giardia lamblia genome sequence released on 1/1/2003 [Dictyostelium discoideum were downloaded from TIGR [The annotated genome sequences of rice  were parsed and manually checked for the presence of the characteristic features of the WRKY domain.We searched 'nr' and dbEST datasets for WRKY genes in species outside the plant phyla. The dbEST dataset was also used to survey the expressed WRKY genes in plant species. We aligned the sequences in the datasets with WRKY-seed using BLAST programs . To deteG. lamblia, we searched their genome sequences with blastp and PSI-BLAST [-4 for blastp and PSI-BLAST and E < 0.1 for HMMER were manually compared to remove non-WRKY hits. We also used the same strategy to identify the set of WRKY genes from the Arabidopsis genome.To systemically catalog the WRKY genes for rice and SI-BLAST  using WRSI-BLAST . HMMER, SI-BLAST , is avaiSI-BLAST . The sea-4) were then subject to WRKY domain and gene predictions. The WRKY domain was predicted with the Pfam's DNA SEARCH [To identify WRKY genes from the green alga, we first BLASTed its genome sequence against the WRKY-seed. The significantly aligned sequences , > 97% for 300 ~ 399 nt, > 98% for 200 ~ 299 nt, > 99% for 100 ~ 199 nt, and = 100% for 50 ~ 99 nt. The alignment with < 50 nt was discarded.We also searched ESTs and EST-assembled contigs for the identified WRKY genes of rice, the green alga, The WRKY domain boundary was defined as by Eulgem et al. . The pepLW initiated the study. YZ and LW carried out the analyses, and YZ drafted the manuscript.Giardia lamblia, Dictyostelium discoideum and Chlamydomonas reinhadrtiiWRKY genes from Click here for fileMultiple alignments, domain classification and sequence conservation patterns of WRKY domains from rice (OsWRKY), Arabidopsis (AtWRKY), the green alga (ChrWRKY), the slime mold (DsWRKY) and Giardia lamblia (GlWRKY)Click here for fileIdentified members of the WRKY superfamily in the rice genomeClick here for fileSurvey of WRKY genes from ESTs or their assembled gene indices for 19 plants and the phylogenetic classification of the genesClick here for file


S. aureus, one of the main etiologic agents of brain abscess in humans, is a potent stimulus for proinflammatory mediator production. Recent evidence from our laboratory indicates that Toll-like receptor 2 plays a pivotal role in the recognition of S. aureus and its cell wall product peptidoglycan by glia, although other receptors also participate in the recognition event. This review will summarize the consequences of S. aureus on CNS glial activation and the resultant neuroinflammatory response in the experimental brain abscess model.Brain abscess represents a significant medical problem despite recent advances made in detection and therapy. Due to the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, the occurrence of brain abscess is likely to persist. Our laboratory has developed a mouse experimental brain abscess model allowing for the identification of key mediators in the CNS anti-bacterial immune response through the use of cytokine and chemokine knockout mice. Studies of primary microglia and astrocytes from neonatal mice have revealed that  The leading etiologic agents of brain abscess are the reported ,2. Brainreported . The mosAt the histological level, brain abscess is typified by a sequential series of pathological changes that have been elucidated using the experimental rodent models described in detail below -7. StagiS. aureus, one of the main etiologic agents of brain abscess in humans. The mouse brain abscess model accurately reflects the course of disease progression in humans, providing an excellent model system to study immunological pathways influencing abscess pathogenesis and the effects of therapeutic agents on disease outcome. We have successfully utilized this model to characterize inflammatory mediators induced in the brain immediately following S. aureus exposure [in vivo [S. aureus leads to the immediate and sustained expression of numerous proinflammatory cytokines and chemokines in the brain including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1α,β, macrophage inflammatory protein-2 (MIP-2/CXCL2), monocyte chemoattractant protein-1 (MCP-1/CCL2), MIP-1α/CCL3, MIP-1β/CCL4, and regulated upon activation T cell expressed and secreted (RANTES/CCL5) [Although case reports of brain abscess in humans are relatively numerous, studies describing the nature of the ensuing CNS and peripheral immune responses are rare. Therefore, our laboratory has developed a mouse experimental brain abscess model to elucidate the importance of host immune factors in disease pathogenesis ,7-9. Ourexposure  as well [in vivo . For exaS. aureus infection persists, effectively augmenting damage to surrounding normal brain parenchyma [S. aureus exposure [S. aureus infection was found to induce a chronic disruption of the blood-brain barrier, which correlated with the continued presence of peripheral immune cell infiltrates and glial activation [As mentioned earlier, lesion sites in both our experimental model and in human brain abscess are greatly exaggerated compared to the localized nature of bacterial growth, reminiscent of an over-active immune response. To account for the enlarged region of affected tissue involvement associated with brain abscesses compared to the relatively focal nature of the initial insult, we have proposed that proinflammatory mediator production following renchyma . Specifiexposure . Concomitivation . CollectBesides the potential detrimental roles cytokines may exert on surrounding normal brain parenchyma during the later stages of brain abscess, numerous proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 may have beneficial effects on the establishment of host anti-bacterial immune responses. These cytokines exert numerous functions within CNS tissues including modulation of blood-brain barrier integrity, induction of adhesion molecule expression on cerebral microvascular endothelial cells, and subsequent activation of resident glia and infiltrating peripheral immune cells -17. We rS. aureus-induced acute cerebritis was demonstrated by Lo et al. where transient neutrophil depletion resulted in enhanced pathology [S. aureus-specific lymphocytes [Neutrophils are potent bactericidal effector cells and represent the major peripheral cell infiltrate associated with developing brain abscesses ,9. Neutrathology . In addiathology ,8. The pathology  that participate in the initiation of the anti-bacterial adaptive immune response remain to be elucidated. Evidence to support the establishment of an adaptive immune response is provided by our recent findings that elopment . It is nS. aureus and its cell wall product peptidoglycan (PGN) serve as potent stimuli for proinflammatory mediator production in primary microglia [S. aureus infection, well before the significant accumulation of peripheral immune cell infiltrates [S. aureus induces IL-1β expression in neonatal rat microglia [Relevant to our experimental brain abscess model, recent studies from our laboratory have established that both intact icroglia ,10,25. Siltrates . Anothericroglia .S. aureus and PGN are capable of inducing microglial MHC class II [S. aureus to augment the expression of receptors that are important for antigen presentation suggests that the ability of microglia to present bacterial peptides to antigen-specific T cells may be greatly enhanced following an initial exposure to S. aureus. The effects of S. aureus and PGN on microglial CD40, CD80, CD86, and MHC class II expression may either be a direct consequence of bacterial stimulation or indirect via the autocrine action of cytokines produced by activated microglia.Microglia represent one of the main antigen presenting cells in the CNS ,27. To aclass II ,25, CD40class II . The abiS. aureus and PGN with significant proinflammatory mediator expression, and many of these same mediators are persistently elevated in brain abscess. Drawing on this relationship, we have proposed that chronic microglial activation may contribute, in part, to the excessive tissue damage characteristic of brain abscess. Therefore, attenuating chronic microglial activation subsequent to effective bacterial elimination in the brain may result in attenuation of the structural and functional damage associated with brain abscess. We have recently examined the efficacy of the cyclopentenone prostaglandin 15d-PGJ2 to modulate microglial responses to S. aureus [2 was found to be a selective and potent inhibitor of S. aureus-dependent microglial activation through its ability to significantly attenuate the expression of numerous proinflammatory cytokines and chemokines of the CC family including IL-1β, TNF-α, IL-12 p40, MCP-1, and MIP-1β. In addition, 15d-PGJ2 also selectively inhibited the S. aureus-dependent increase in microglial TLR2, CD14, MHC class II, and CD40 expression whereas it had no effect on the co-stimulatory molecules CD80 and CD86. The ability of 15d-PGJ2 to modulate the expression of these receptors may serve as a means to regulate microglial and T cell activation during gram-positive bacterial infections in the CNS. Preventing microglial activation by 15d-PGJ2 or related compounds may help to resolve inflammation earlier, resulting in reductions in brain abscess size and associated damage to surrounding normal brain parenchyma.Microglial activation is a hallmark of brain abscess ,5,9. The. aureus . 15d-PGJS. aureus and respond with robust production of numerous proinflammatory mediators. However, to date, the receptor repertoire responsible for bacterial recognition remains to be defined. In macrophages, numerous receptors have been implicated in bacterial phagocytosis and subsequent activation leading to proinflammatory mediator release including Toll-like receptors (TLR), scavenger receptors, and mannose receptors. The fact that microglia and macrophages share many functional and phenotypical characteristics supports the contention that these receptors may play an important role in microglial responses to bacteria.As detailed above, our laboratory has established that microglia are capable of recognizing S. aureus and PGN lead to significant increases in TLR2 mRNA and protein expression, which may enhance microglial sensitivity to bacteria during the course of experimental brain abscess development [S. aureus (Kielian, manuscript in preparation). These findings indicate that an alternative receptor(s) is involved in mediating responses to intact bacteria. Candidates include the mannose receptor and members of the scavenger receptor family.Toll-like receptors are a family of surface receptors expressed on cells of the innate immune system that allow for the recognition of conserved structural motifs on a wide array of pathogens (referred to as pathogen-associated molecular patterns) ,33. To delopment . Recent S. aureus [S. aureus and PGN differentially modulate the expression of several distinct scavenger receptors that may influence the nature and extent of phagocytosis (Kielian, unpublished observations). Scavenger receptors have been implicated in β-amyloid phagocytosis by microglia in the context of Alzheimer's disease, in part, by the finding that microglia associated with senile plaques express a high degree of scavenger receptor immunoreactivity [S. aureus phagocytosis by microglia remains to be established.Scavenger receptors encompass a broad range of molecules involved in receptor-mediated phagocytosis of select polyanionic acids such as lipoteichoic acid of . aureus . Althoug. aureus . In the . aureus . Prelimiactivity ,43. In aactivity -47. The S. aureus exposure, suggesting that the regulation of mannose receptor expression is conserved among diverse stimuli [S. aureus exposure remain to be defined. In addition to the receptors described above, there are additional candidates that may serve as receptors for S. aureus phagocytosis in microglia including complement receptor 3  and CD14, the latter of which we have shown to be expressed on microglia and significantly upregulated following activation with either S. aureus or PGN [Microglia have been shown to express functional mannose receptors that are responsible for the binding and phagocytosis of mannosylated and fucosylated ligands of bacteria ,49. Inte stimuli . Followi stimuli -52. In a stimuli . The funs or PGN ,25.Astrocytes play a pivotal role in the type and extent of CNS inflammatory responses. These cells likely play an important role in the initial recruitment and activation of peripheral immune cells into the CNS during neuroinflammation through the production of several cytokines and chemokines, such as IL-1, IL-6, IL-10, TNF-α, IFN-α/β, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), transforming growth factor-beta (TGF-β), RANTES, MCP-1, and IFN-γ-inducible protein-10 (IP-10/CXCL10) ,54.S. aureus and PGN and that they respond with vigorous proinflammatory cytokine and chemokine production [S. aureus-activated astrocytes are NO, TNF-α, IL-1β, MIP-2, MCP-1, MIP-1α, and MIP-1β. These proinflammatory chemokines may serve as signals for neutrophil (MIP-2), monocyte and lymphocyte (MCP-1, MIP-1β) recruitment in vivo, whereas IL-1β and TNF-α likely alter blood-brain barrier permeability and induce the expression of critical adhesion molecules on CNS vascular endothelium required for immune cell extravasation into brain abscesses.Various studies have documented the ability of LPS to induce nitric oxide (NO), cytokine, and chemokine production in astrocytes ,56. In coduction . Among tS. aureus and PGN and in subsequent cytokine and chemokine expression by astrocytes [S. aureus. For example, TLR2 has been shown to form functional heterodimers with TLR1 and/or TLR6 [S. aureus and PGN is currently not known.Astrocytes have recently been shown to express TLR2 ,58, and trocytes . Interes/or TLR6 ,60, ther/or TLR6  and enha/or TLR6 -64. Rece/or TLR6 ,65. Alte/or TLR6 -68 may bS. aureus [S. aureus, indicating that bacterial uptake occurs via a phagocytic pathway and is not simply the result of productive infection by live organisms. Interestingly, TLR2 is not a major receptor for bacterial phagocytosis in astrocytes since both TLR2 KO and wild type astrocytes were equally capable of phagocytosing intact S. aureus organisms in vitro [S. aureus phagocytosis by astrocytes and the optimal conditions required for bacterial uptake are currently ongoing in our laboratory. Issues such as whether bacterial internalization is serum-dependent or requires other bacterial binding proteins must also be addressed.Although astrocytes have been shown to possess phagocytic activity in response to β-amyloid , apoptot. aureus . An actiin vitro . The recS. aureus experimental brain abscess model provides an excellent tool for deciphering the importance of various mediators in disease pathogenesis. Especially appealing is the ability to examine the role of specific factors using transgenic and knockout mice because, in our experience, all of the mouse strains examined with this model have qualitatively similar inflammatory profiles following bacterial challenge. In addition, the consequences of S. aureus infection do not appear to be influenced by gender, as the responses of female and male mice are similar- another advantage when performing studies with knockout or transgenic mice where animal numbers are often limiting.The incidence of brain abscess is expected to persist in the human population due to the ubiquitous nature of bacteria coupled with the recent emergence of antibiotic-resistant bacterial strains. Therefore, understanding the roles of both host anti-bacterial immune responses along with bacterial virulence factors may lead to the establishment of novel therapeutic treatments for brain abscess. The mouse S. aureus have been elucidated in terms of proinflammatory mediator expression and in general, have been found to be qualitatively similar to those observed following LPS exposure. Although studies with primary microglia and astrocytes from TLR2 KO mice reveal an important role for this receptor in mediating S. aureus-dependent activation, it is clear that additional receptors are also involved in glial responses to this bacterium. This functional redundancy is not surprising because these pathogens have the potential for devastating consequences in a tissue that has limited regenerative capacity such as the CNS.The responses of microglia and astrocytes to S. aureus bactericidal activity in vitro, suggesting that they may also participate in the initial containment of bacterial replication in the CNS. However, their bactericidal activity in vitro is not comparable to that of neutrophils or macrophages, suggesting that this activity may not be a major effector mechanism for microglia during acute infection. Third, activated microglia have the potential to influence the type and extent of anti-bacterial adaptive immune responses through their upregulation of MHC class II and co-stimulatory molecule expression. Finally, if glial activation persists in the context of ongoing inflammation, the continued release of proinflammatory mediators could damage surrounding normal brain parenchyma. Indeed, inappropriate glial activation has been implicated in several CNS diseases including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis as well as Alzheimer's disease. The continued use of transgenic and knockout mice for in vivo studies will facilitate our understanding of immune mechanisms contributing to brain abscess pathogenesis.The implications of glial cell activation in the context of brain abscess are likely several-fold. First, parenchymal microglia and astrocytes may be involved in the initial recruitment of professional bactericidal phagocytes into the CNS through their elaboration of chemokines and proinflammatory cytokines. Second, microglia exhibit BBB blood-brain barrierCCL CC chemokine ligandCD cluster of differentiationCSF cerebral spinal fluidCXCL CXC chemokine ligandCXCR CXC chemokine receptorGFAP glial fibrillary acidic proteinGM-CSF granulocyte-macrophage colony-stimulating factorIFN interferonIL interleukinIP-10 interferon-inducible protein-10KO knockoutLPS lipopolysaccharideM-CSF macrophage colony-stimulating factorMCP monocyte chemoattractant proteinMHC major histocompatability complexMIP macrophage inflammatory proteinNO nitric oxidePGN peptidoglycanRANTES regulated upon activation T cell expressed and secretedTGF transforming growth factorTNF tumor necrosis factorNone declared.


Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines.Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used.This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations. Endogenous reference alse referred to as house keeping genes defines in biology the theoretical assumption that certain genes are ubiquitously expressed in nucleated cells possibly because their stable expression is essential for cell survival and welfare in all physio-pathological circumstances. In practical terms, endogenous reference genes provide a useful constant reference to normalize the expression of test genes in different tissues and in different conditions. This is obviously important when estimates of gene expression are provided in relative terms rather than absolute units of measurement. Thus, endogenous reference genes are used as common denominator in biological fractions where the expression of a test gene is described as the relative ratio over an arbitrarily selected internal control presumed to be stably expressed in all circumstances relevant to the experiment -3. Most Powerful insights in patterns of gene expression could be attained recently through cDNA or oligonucleotide-based global transcript analysis tools that apply a constant reference system to determine ratios of gene expression across large data sets ,29. The Reproducibility assessment of the data set through determination of reference concordance. This was achieved by repeating 14 reference experiments using the melanoma cell line A375 as test sample (Cy5) co-hybridized with pooled PBMC as reference (Cy3). To test for inter-array and printing variation, slide number one and every other 25 slides in sequential order of printing (100 slides per printing set) were used for the repetitive A375 / pooled PBMC hybridizations. In addition, to assess labeling bias, reciprocal labeling was alternatively applied as previously described [Identification of putative endogenous reference genes was performed on 384 array experiments of unequivocal quality by selecting genes that had demonstrated high reference concordance (>90% of the genes in the arrays) and ranking them from the lowest to the highest variance of Log2 test / reference ratios across all array experiments. 3) Validation of the candidate endogenous reference genes as predictor of relative gene expression in large data series. For this purpose, we tested the relative estimates of expression of the melanocytic lineage-restricted melanoma differentiation antigen gp100/Pmel17 (gp100) [Validation of endogenous reference gene-based normalization of gene expression by qRT-PCR and protein expression.In the present study, we tested a set of 419 consecutive experiments performed on a 17,000 gene cDNA array platform to which RNA from neoplastic or normal tissues were consistently co-hybridized with a differentially-labeled reference RNA derived from peripheral blood mononuclear cells (PBMC) pooled from six normal donors. The following steps were pursued: 1) escribed . In this (gp100)  in melan. Thirteen arrays with missing data in more than 30% of the spots and 8 arrays with irregular distribution according to M-A plots (M and A representing respectively log-ratios and average intensities) [log2Cy5/Cy3 ratios were normalized by approximating median values to zero. Spots with ratios > 100 or < 1/100 were truncated at 100 and 1/100 respectively. Data normalization was done by median centering Log2ratio of all genes for each array. For data normalization we used certain arbitrary cutoff criteria to remove spots with weak signal. Weak signal approximating background fluorescence is not reliable and the corresponding log ratios are often distorted resulting in disproportionately high or low values that would bias the statistical results. Because the interpretation of spots with low signal is difficult to make we have adopted the policy of excluding them to focus the analysis on genes whose expression pattern is more reliably tracked by the array tool. In regard to spots with a ratio of >100 or <-1/100, most of them are due to extremely weak signal in one channel which generates a disproportionately extreme value. Although there is no published "gold standard" for the selection of such cutoffs, in practice the parameters that we used for this study are most commonly accepted by investigators and reasonable because allow retention of most of the data in the array excluding only the most extreme and least reliable information.The GenePix Pro 4.0 software was used for array image analysis and calibration. All statistical analyses were performed with the SPLUS package nsities)  were exct test as those with ratios significantly different between the 7 forward and 7 reciprocal experiments after reversal of the reciprocal values (P < 0.05) and the median ratio difference between the 7 forward and 7 reciprocal experiments was larger than 1.5 fold. The genes whose variances of the log ratio across all experiments were among the top 1 percentile of variance of all genes were identified as discordant due to hybridization bias. In addition, genes with more than 50% missing values were excluded. Overall 1,343 out of 16,738 were judged potentially discordant and were excluded from further analysis. The remaining 15,395 concordant genes were used for subsequent analyses.To select from the data base the most reliable genes, we first assessed reproducibility by testing the level of concordance in 14 reference experiments. Reference concordance relies on the expectation that results obtained by repeatedly hybridizing the same test and reference material should perfectly collimate and the degree of deviation from such prediction estimates experimental variance. Concordance can be easily measured by periodically re-hybridizing the same test sample with the constant reference sample. We analyzed a matrix of 7 forward and 7 reciprocally-labeled replicate array experiments hybridized periodically every other 25 cDNA array slides. Reciprocal labeling was applied to measure labeling bias . Data geWe studied 384 of 419 consecutive array experiments remaining after the exclusion of the 14 reference concordance experiments and 21 experiments judged of poor quality due to missing data or irregular distribution by M-A plot analysis. Selection of candidate endogenous reference genes followed these steps.log2 ratio). Consequently, genes with fold changes < 1.46 from zero across all 384 arrays were categorized as candidate endogenous reference genes and were ranked according to ascending values of SD of Log ratio  were used to calculate experimental variance Table . Median Log2 ratio had to be within 2 SD from zero and the average intensities of both channels across all samples need to be higher than 2,000. The intensity parameter was added to ensure that the selected endogenous reference genes were expressed at a relatively high level in most tissues to mitigate excessive fluctuations in Log2 ratios occurring when a low value is applied as denominator. This is important when a reference gene is applied as a denominator in the equation used to normalize the ratio of other tests genes; in such cases robust gene expression decreases the range of ratios resulting from the analysis decreasing, therefore, the experimental variance. Sixty-nine genes fit these criteria. The range of mean Log2 ratio was from 0 to 0.15 and the SD of mean Log2 ratio was from 0.27 to 0.42. Analysis of 7 replicate array based on the same group of candidate endogenous reference genes demonstrated a similar distribution of mean Log2 ratio and SD of mean Log2 ratios (with exception of TFAP2B) suggesting that this variance could be attributed to predominantly experimental noise. Among these genes we further selected for validation 11 that had high mean fluorescence intensity  were found errors in endogenous reference gene selection ."Leave-one-out cross-validations" were used to estimate the error ,34. In e. We found a good correlation between the expression patterns observed by us and that reported by the Affymetrix GeenChip array (data not shown). In addition, a cluster of endogenous reference genes suggested by Applied Biosystems for standardization during qRT-PCR was analyzed by evaluating the SD of their Log2 ratios across the 384 arrays. As data indicated in Table Log2 ratios comparable to that of the endogenous reference genes identified in this study, the corresponding percentage of the arrays in which Log2 ratios were within 2 SD from zero ranged between 0.68–0.85 which is significantly lower than the frequency in which the same parameter fell for the 69 candidate house keeping genes identified here (>95%). In addition, we looked at the variation in our arrays of genes classically used as endogenous reference genes including: ribosomal protein L32, HRPT, β-actin and tubulin-α3. In all cases the mean SD of Log2Ratio was relatively larger than for the endogenous reference genes identified in Table The expression of each candidate endogenous reference gene identified in this study was then compared with available information about the expression of the same genes in 12 normal human tissues reported by the Affymetrix HG-U95A-E probe sets To validate the usefulness of candidate endogenous reference genes in large sample populations we developed a new tool that we are planning to use in the future for validation of gene expression across extensive data sets. This tool displays cDNA libraries originated from different tissues or cell lines individually spotted on a solid surface. The principle of this technology is similar to RNA dot blot which uses RNA isolated from samples and transferred to membrane making, therefore, the transcriptome array a high-density dot blot. The labeled DNA probe of interest is hybridized to the immobilized complementary strain of mRNA. A reference gene hybridization will carried out simultaneously to estimate the relative expression of the gene of interest compared with the reference gene. The new tool we describe here, termed transcriptome array, utilizes cDNA generated from source mRNA for target immobilization to improve the stability of the immobilized targets and differentially fluorescence-labeled test and reference probes (RNA or double strained DNA) then can co-hybridize on to the same spots. Using a validated RNA amplification technology , large qTo test the usefulness of various endogenous reference gene we resorted to the well characterized expression of the melanocytic lineage-specific gene gp100/Pmel17  that is Log2 Cy5 / Cy3 ratios between melanocytic and non-melanocytic tissues. gp100 was, as expected, expressed more in melanocytic lesions with high degree of significance no matter what gp100 / endogenous reference gene combination was used. The range of significance, however, varied extensively depending upon the gp100 / endogenous reference gene combination. This difference was considered representative of the ability of different endogenous reference genes to normalize for tissue-specific gene expression patterns . There was, however, a big range in the discriminatory capacity among endogenous reference genes with NEDD8, RAB3 and FZD6 providing the highest predictive value  and β-actin being one of the least reliable .As endogenous reference genes we chose β-Actin and new candidate genes identified by this study (see previous section). The test genes (gp100, Rho-C and DICE) were separately hybridized to the transcriptome array. Each gene labeled with Cy3 was co-hybridized with individual endogenous reference genes labeled with Cy5. We then compared Log2 ratios obtained with all endogenous reference genes and those obtained using the genes that provided the best five or the best three results . The basis for this test was that non-melanocytic lesions uniformly should not express gp100 and, therefore, the Log2 ratios for any gp100 / endogenous reference gene combination should be constant resulting in very low SD. SD for non-melanocytic lesions were much lower using pooled endogenous reference genes for the normalization . Similar results were obtained when results from the best 5 and best 3 endogenous reference genes were pooled together. In addition, even if pooling data together did not provide the highest level of discrimination compared to the best results obtained with selected individual endogenous reference genes, the capacity of pooled data sets to discriminate gp100 expression between melanocytic and non melanocytic samples was still very high . Interestingly, different endogenous reference genes provided not only different levels of discrimination but also provided different estimates of gp100 expression in melanocytic lesions with gp100 / endogenous reference gene Log2 ratio above (NEED8, RAB3, FZD6, AFAP, SNRPD3, HIRIP5, GPLD1) or below (COL8A1, β-actin) 0  the discriminatory capacity of endogenous reference genes in samples with known patterns of expression of gp100. We selected 6 previously characterized ,36 melanAlthough the concept of endogenous reference genes is appealing it may be unrealistic to expect that a gene could be equally expressed in all eukaryotic cells in all physiopathological conditions. Thus, the endogenous reference gene concept can only approximate such ideal biological behavior. In fact, numerous studies noted that the expression of the ostensible endogenous reference genes varies according to distinct physio-pathological situations and can be affected by experimental manipulation ,26,27,41In this study, therefore, we looked for relative stable endogenous reference gene challenging a 17,000 cDNA clone data base against approximately 400 specimens that included a broad variety of normal and neoplastic tissues as well as cell lines. Indeed, few previously unnamed genes approximated the ideal endogenous reference gene by being expressed consistently in more than 95% of the specimens analyzed within a variance that was barely and insignificantly different from what could be expected from intrinsic experimental fluctuation . However, gene expression analysis is done to determine such specificity and in most cases no previous knowledge of tissue specificity is truly available. One may argue that, in spite of the extreme variability in normalization power demonstrated by various endogenous reference genes, still significant differences could extracted between the melanocytic and non-melanocytic samples no matter what endogenous reference genes were used. This statement is correct in this experimental situation where hundreds of samples could be compared with the transcriptome array. However, in cases when only few samples are analyzed the difference in discriminatory capacity becomes critical. For instance, when randomly selected gp100 / endogenous reference gene The lowest limit of detection in the transcriptome array of specific probes in term of copy numbers is currently under investigation. Decreasing concentrations of β-actin alone printed the array spots as internal controls suggest that the detection range is from 30 ng / spot to 3 pg / spot when standard fluorescent intensity range parameters are applied. The higher limit of detection before saturation can be easily adjusted to avoid under estimates of highly expressed genes, therefore, increasing the dynamic range of detection. This is achieved by adjusting the fluorescent intensity right below the saturation for both channel and subsequently calculating the abundance of reference gene expression using serially diluted internal control spots as standard curve. With few exceptions, the expression of most genes can be estimated with relative accuracy using this semi-quantitative approach.Since in most cases endogenous reference genes are selected based on previous knowledge of their usefulness for a particular experimental situations, it is likely that the best consistency could be achieved in such circumstances when averages of several endogenous reference gene normalizations are used. This may represent a reasonable solution particularly if a cocktail of endogenous reference genes identically labeled could be used as reference within a single experiment. We are presently testing various combinations of lineage specific genes and pools of endogenous reference genes for hybridization to the transcriptome array and for qRT-PCR. Such attempts, however, have been quite disappointing so far as results obtained using mixture of endogenous reference genes do not yield information comparable to that achievable when results obtained with the same endogenous reference genes used individually are pooled together. This is possibly due to imbalanced density of various endogenous reference genes in different samples with predominant effects of some over others. Therefore, extensive work in the future will be aimed at equilibrating the interactions among endogenous reference genes in various experimental conditions to test whether mixtures of them could be used in multiplex for normalization of large expression analysis studies.Our observations, together with previous work by others indicate that much caution should be taken when using endogenous reference to normalize the expression of test genes. It is likely that previous analyses based on single endogenous reference genes might have been strongly biased by the individual selections and the interpretation of the results should looked at with caution. In particular, this study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference genes, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.The list of samples used for cDNA microarray hybridization or for preparation of the transcriptome array is available upon request. Most melanoma and renal carcinoma cell lines were generated at the Surgery Branch, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD and maintained in RPMI  supplemented with 10% fetal calf serum (Biosource). Cell lines were harvested by Trypsin /Versene (Biosource) digestion. PBMC were collected by leukapheresis from six unrelated normal donors in the Department of Transfusion Medicine, Clinical Center, NIH and purified by Ficoll gradient separation. Surgically removed tumor tissues were collected from the Tissue Network (Philadelphia, PA), Surgery Branch and CPSB specimen bank, NCI, (Bethesda, MD). Fine needle aspiration (FNA) biopsies were obtained from patients with metastatic melanoma referred to the Surgery Branch, NCI for immunotherapy. Total RNA was isolated using Qiagen RNeasy kit and its quality and quantity was estimated using Agilent Bioanalyzer 2000 .in vitro into anti-sense RNA (aRNA) and reverse-transcribed into fluorescence labeled cDNA for hybridization to 17,000 gene cDNA-based microarrays [Total RNA extracted from test and reference samples was transcribed roarrays ,44. Pool.cDNA (UniGene cluster) microarrays were printed at the Immunogenetics Section, DTM, CC, NIH with a configuration of 32 × 24 × 23 containing 17,500 elements. Clones used for printing were selected from the Research Genetics RG_HsKG_031901 8 k clone set and the 9k RG_Hs_seq_ver_070700 40 k clone set. The 17,500 spots included 12,072 uniquely named genes, 875 duplicate genes and about 4,000 expression sequence tags. For a complete list of genes included in the Hs-CCDTM-17.5k-1px printing please visit our web site at .A collection of aRNA-based libraries was prepared from 960 frozen tissue samples or cell lines and individual aRNAs were reverse transcribed into cDNA in the presence of 1 μl of dN6 primer (8 μg/μl), 6 μl of first strand buffer, 3 μl of 10 mM dNTP, 3 ul of 50 mM dTT, 1.5 μl of Rnasin and 10 μl of aRNA (6–12 ug) with addition of 3 μl of Superscript II (BRL) in 5.5 μl volume after heating to 65°C for 5 minutes. Reactions were carried out in 96-well plates at 42°C for 90 minutes followed with addition of 1 μl Rnase H and heating for 20 min at 37°C. cDNA were further purified by utilizing CentriCept gelfitration plates to remove non incorporated primer and dNTP. Purified cDNA were transferred to 384-well plates and dried by speedvac. Samples were re-suspended in 13 μl of 3 × SSC followed by shaking at 3,000 rpm for 15 min. Reconstituted cDNA libraries from individual samples and spiked reference gene at different concentration were printed on to poly-L-lysine coated glass slides at the concentration of 0.5–1 μg/μl using the OmniGrid (GeneMachine. San Carlos, CA) printer and Telechem printing pins . Each 100 μm diameter spot was duplicated at a 250 μm distance. After complete exsiccation, slides were post-processed as described at . In order to generate single strand fluorescence labeled cDNA for hybridization, modified specific 5' primers with an extension of the T7 promoter region were used for PCR amplification. Double strand PCR fragments (300–800 bp) were used as template for in vitro transcription to generate single stranded mRNA. 3 μg of amplified RNA were then reverse transcribed into cDNA in present of 4 μl of first strand buffer, 1 μl dN6 primer , 2 μl 10 X low T-dNTP (5 mM A, C and GTP, 2 mM dTTP), 2 μl Cy-dUTP (1 mM, Cy3 or Cy5), 2 μl 0.1 M DTT, 1 μl RNasin, 3 μg amplified mRNA in 8 μl DEPC H2O. After heating to 65°C for 5 minutes and cooling to 42°C, 2 μl of SSII was added and the labeling reaction was carried out at 42°C for 90 min. Probe purification and hybridization were performed as previously described [Specific PCR primers for the amplification of each gene probe were designed using Primer 3 program ragments 0–800 bp escribed .max = 518 nm) and at the 3' end with the quencher dye molecule 6-carboxytetramethyl-rhodamine . Total RNA was used for gene expression validation. Measurement of gene expression was performed using the ABI 7900HT sequence detection system (Perkin Elmer, Foster City, CA) as previously described [Primer and probe sets from each candidate genes were designed using the Primer Express 2.0 program (Applied Biosystems, Foster City, CA) and synthesized by BioSource. Taqman probes were labeled at the 5' end with the reporter dye molecule 6-carboxy-fluorescein (FAM; emission λescribed ,45. Messescribed .PJ validated the system combining high-throughput data with gene-directed analysis by quantitative real-time PCR. YZ performed statistical analysis. YN, MCP, DN, VM and KS participated in the preparation and utilization of array experiments used for the identification of stably expressed genes. NH, HS and PRT provided a large library of samples necessary for the development of the transcriptome array. FMM supervise the project as principal investigator.EW provided leadership for the development of cDNA arrays, transcriptome array and for the overall conduct of the experiments.


Drosophila immune response involves three types of hemocytes (‘blood cells’). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. Here, we have investigated the mechanisms underlying the specification of lamellocytes. We first show that collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. We therefore propose that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells. The lamellocyte is induced to differentiate under conditions such as parasitization by wasps and is shown in this study to require collier, the orthologue of the vertebrate early B-cell factor Drosophila shares several features with the analogous process in vertebrates. A first population of embryonic hemocyte precursors (prohemocytes) is specified from the head mesoderm very early during embryogenesis. At the end of larval stages and the onset of metamorphosis, a second population of hemocytes is released from a specialised hematopoietic organ, the larval lymph gland (Hematopoiesis in ph gland . Both poph gland . Strikinph gland . Howeverph gland , the mecph gland .collier (col) is expressed in the lymph glands at the end of embryogenesis  (Drosophila. On the basis of Col expression and col mutant phenotypes, we propose that this factor confers an instructive function on a discrete subpopulation of cells in the Drosophila definitive hematopoietic organ.During our search for genes involved in specification of lamellocytes, we observed that ogenesis . The genor (EBF) , a key for (EBF) . We showDrosophila lymph glands at the end of embryogenesis  are lethal at the late embryonic stage  parasitization by producing lamellocytes. This dedicated cellular response is maximal in wild-type (wt) larvae 48 h after wasp egg-laying 06949 AN293 06949 . Whereas1 larvae D and 2E.2R)AN293 . In no cestation , col1 ise33C-Gal4 line, which drives expression in lymph glands   , with a pen-lacZ . Around pen-lacZ G, which pen-lacZ . This phpen-lacZ . Thus, wal cells , consistal cells . No prod lz-Gal4  and hml- drivers , respectAt the end of larval stages, the lymph gland is composed of four to six paired lobes. The two anterior (primary) lobes that formed in the embryo  contain Ser mutant cells in the larval lymph glands further indicated that Ser-expressing cells are responsible for activation of Lz expression in surrounding cells and their commitment to a crystal cell fate (Col expression in a posterior cluster of cells of the primary lobes is reminiscent of that of Serrate (Ser), a Notch ligand . The Serell fate . Togetheell fate F–3H. Howell fate   Col expression defines a specific group of cells within the lymph glands; (ii) lamellocyte differentiation, which is an exclusive feature of lymph gland hematopoiesis, depends upon Col activity; and (iii) the massive production of lamellocytes that follows parasitization does not involve changes in Col expression. We thus propose a two-step signalling model for induction of lamellocytes in response to wasp egg-laying A. AccordDrosophila cellular immune response, and EBF function in B-cell development in vertebrates, suggests that Col/EBF function was co-opted early during the evolution of cellular immunity. A puzzling question remains, however, of how the cell-autonomous function of EBF in B-cell development, and the non–cell-autonomous function of Col in lamellocyte development, could relate to an ancestral Col/EBF function. We would like to propose that the ancestral expression of Col/EBF in a subset of hematopoietic cells conferred on these cells the ability to respond to signals from circulating immune supervisors (generically designated as macrophages in Drosophila PSC on the other. Although admittedly highly speculative, this proposal takes into account the following considerations. B-cell development represents the default fate of lymphoid progenitors (Pax5. The −/−Pax5 pro–B-cells retain the ability to generate a whole range of both ‘innate’ myeloid and lymphoid cells (Pax5, acting downstream of EBF, for which there is no known counterpart in Drosophila hematopoiesis. Second, the co-opting of Col activity for lamellocyte differentiation in larval hematopoiesis most likely came on top of a preexisting hematopoietic system, such as that operating in Drosophila embryos (B- and T-lymphocytes mediate adaptive immunity, a phylogenetically recent component of the immune system as it is found only in gnathostomes . How adagenitors . Althougid cells . Thus, t embryos . FurtherL. boulardi for 2–4 h, then allowed to develop at the appropriate temperature and analysed 24 or 48 h later. Hemocyte observation and counting, and lacZ staining of lamellocytes, were as previously described (Unless otherwise stated, all fly stocks were maintained at 25 °C on standard medium, and genotypes were verified with marked balancer chromosomes. For wasp infection, second instar larvae were submitted to egg-laying by escribed .srpD-Gal4 transgene: A distal promoter fragment, between 8.8 and 6 kb upstream of the srp transcription start site and a 340-bp fragment overlapping this site were amplified by PCR using 5′-GCTAGCGACGCGTGATGCAACTTAATCAA-3′ and 5′-CTGCAGTTTATGAATGGAAGACGCGGACG-3′ primers, and 5′-CTGCAGACGGCCAAGTCCAACAACAACAA-3′ and 5′-GGATCCCTGTTGCTGCTGTAACTGTTGAT-3′ primers, respectively, then fused before subcloning upstream of the Gal4 coding sequence in a pCaSpeR vector. Transgenic lines were obtained by standard procedures. Because they are embryonic-lethal at 25 °C, the srpD-Gal4/UAS-col animals were kept at 18 °C before shifting to 25 °C at the second larval instar.The Immunostaining and in situ hybridisation of larval lymph glands and embryos were performed as in