We conducted this analysis to determine i) which journals publish high-quality, clinically relevant studies in internal medicine, general/family practice, general practice nursing, and mental health; and ii) the proportion of clinically relevant articles in each journal.ACP Journal Club for internal medicine and its subspecialties; Evidence-Based Medicine for general/family practice; Evidence-Based Nursing for general practice nursing; and Evidence-Based Mental Health for all aspects of mental health). Original studies and review articles were classified for purpose: therapy and prevention, screening and diagnosis, prognosis, etiology and harm, economics and cost, clinical prediction guides, and qualitative studies.We performed an analytic survey of a hand search of 170 general medicine, general healthcare, and specialty journals for 2000. Research staff assessed individual articles by using explicit criteria for scientific merit for healthcare application. Practitioners assessed the clinical importance of these articles. Outcome measures were the number of high-quality, clinically relevant studies published in the 170 journal titles and how many of these were published in each of four discipline-specific, secondary "evidence-based" journals , four titles supplied 56.5% of the articles and 27 titles supplied the other 43.5%. For Evidence-Based Medicine , five titles supplied 50.7% of the articles and 40 titles supplied the remaining 49.3%. For Evidence-Based Nursing , seven titles supplied 51.0% of the articles and 34 additional titles supplied 49.0%. For Evidence-Based Mental Health , nine titles supplied 53.2% of the articles and 34 additional titles supplied 46.8%. For the disciplines of internal medicine, general/family practice, and mental health , the number of clinically important articles was correlated withScience Citation Index (SCI) Impact Factors.We evaluated 60,352 articles from 170 journal titles. The pass criteria of high-quality methods and clinically relevant material were met by 3059 original articles and 1073 review articles. For Although many clinical journals publish high-quality, clinically relevant and important original studies and systematic reviews, the articles for each discipline studied were concentrated in a small subset of journals. This subset varied according to healthcare discipline; however, many of the important articles for all disciplines in this study were published in broad-based healthcare journals rather than subspecialty or discipline-specific journals. Evidence on the journal-reading habits of clinicians comes from three separate groups of publications. First, several surveys have been used to ascertain the reading habits of physicians. Fafard and Snell  assessedA second set of surveys and studies center on general information-seeking behaviors of clinicians. These studies show how journal reading fits in with the other types of information that clinicians use. Two systematic reviews have been done recently.Researchers at the Australian National Institute of Clinical Studies  reviewedThe third source of information on clinicians' use of information resources comes from marketing studies. The Association of Medical Publications  monitorset al. [The importance of reading journal articles for clinical care is evident. The increasing number of journals from which important and relevant articles are found, combined with the decreasing number of personal subscriptions , makes iet al.  sought tet al. [Cochrane Database for Systematic Reviews for 1997, as well as policy statements from the American Academy of Pediatricians and the Canadian Paediatric Society, they determined that four general medical journals and three pediatric specialty journals provided access to most of the important advances: Archives of Diseases in Childhood, BMJ, JAMA, Journal of Pediatrics, Lancet, New England Journal of Medicine, and Pediatrics. Their results validate the findings of Weiner et al.–important studies in a discipline or specialty are often not published in specialty journals.Since the publication of the study by Weiner et al. , severalet al.  assessedGehanno and Thirion  used MEDet al. [Lee et al.  sampled et al.  were preet al. [et al. provided results of a hand search of 85 core journals of interest to family/general practice. Physicians read these journals for six months and identified articles that were considered to be POEMs (patient-oriented evidence that matters). A POEM addresses a clinical question encountered by a family physician at least once every two weeks, measures patient-oriented outcomes, and presents results that will likely affect practice. The report provides summaries of which journals publish important clinical advances for general/family practice.Ebell et al.  as well et al.  present et al. [In this article we report on a survey of the contents of 170 core clinical journals for the publishing year 2000 to assess which journals publish the highest number of methodologically sound and clinically relevant studies in the disciplines of internal medicine, general/family practice, general practice nursing, and mental health. In the "Methods" section we describe our two-step article selection process for clinical importance and methodologic rigor, which is very similar to that used by Ebell et al. . The datACP Journal Club (ACP J Club) to support internal medicine , Evidence-Based Medicine (EBM) to support general/family practice, Evidence-Based Nursing (EBN) to support general care nursing, and Evidence-Based Mental Health (EBMH) to support mental health. To identify potential candidate articles for inclusion in these journals, six Masters-level trained staff read each article in the major general healthcare journals and those in the disciplines and subdisciplines related to the content of each abstract journal. The list of these journals , except for qualitative and cost studies. Basic inclusion criteria are that the articles i) are about the healthcare of humans; ii) have at least one clinically important outcome; and iii) use appropriate statistical analyses. As an example of category-specific criteria, an article on screening or diagnosis must meet these additional criteria:Original and review articles are placed in one or more of seven categories of study type–therapy and prevention, screening and diagnosis, prognosis, etiology and harm, economics and cost, clinical prediction guides, and qualitative studies . All cat• a spectrum of participants were included, some with the disease or condition of interest and some without• objective diagnoses were made using the "gold" standard or current clinical standard for diagnosis of the disease or condition• participants received both the new test and some form of the diagnostic standard• the diagnostic standard was interpreted without knowledge of the test result and vice versa.The basic inclusion criteria are based on study design and methodology principles for evidence-based healthcare. Their use identifies studies that have data related to patients or those at risk of disease, diseases and conditions, and real-life clinical settings. Therefore, a study or review article that meets the criteria can be considered to be appropriate for possible use in patient care decision-making. The article readers are trained and retested annually so that they can reliably apply these selection rules for inclusion in our evidence-based journals  .With a research grant from the U.S. National Library of Medicine we intensified our data collection from the reading process related to the evidence-based journals for the publishing year 2000. All articles in 170 journals were classified as to whether they were "of interest" to the healthcare of humans and, if so, whether they reported original data or were systematic review articles. These original studies and reviews were classified into all possible categories (where more than one category could apply, for example, a therapy article that included economic data), and were then given a pass or fail methodologic designation for each category. for a copy of the rating system that was used in paper format for this study) some articles were further processed. The editors chose articles to be abstracted that they considered to have the most important message for clinicians. The remaining pass articles were listed as "Other Articles Noted" if their content was of relevance to the disciplines covered by the abstract journals.Articles passing methodologic criteria were assessed further for clinical interest by an editorial group of practicing clinicians for each abstract journal. These clinicians have expertise in methodology and specific areas of healthcare such as gastroenterology or neonatology nursing. At this point the clinician raters excluded all studies with preliminary results, interventions that were not readily available or proven useful, already known and applied findings, and topics addressing rare conditions or diseases. After review, often by a team of three-five clinicians (see ACP J Club, EBM, EBN, and EBMH) to ascertain which journal titles were most important to their target clinical audience . SCI Impact Factors were collected for each journal title for each discipline. If an SCI Impact Factor was not available we sought Social Science Index Impact Factors. These data were analyzed to determine if Impact Factors were related to yield of clinically important advances, as found by Lee et al. [This dual selection process  provided insight into which journals yielded the highest numbers of pass articles. This had major implications for clinical practice at two levels of clinical relevance. The more stringent level includes articles that were summarized in each abstract journal. The second, less stringent level includes articles that are abstracted as well as those articles that are listed in the Other Articles Noted sections. Analysis was done by abstract journal title  Generally, a clinical reader would need to read in the range of 13–14 articles from these top 20 journals to obtain one that is directly clinically important in any healthcare area, although the range is substantial (1.1 to 36.9). We call this number the "number of articles needed to be read" or NNR.The top 20 journals for yield of pass articles are included in Table P = 0.24). Analysis of the top 50 titles showed a weak correlation (correlation coefficient 0.41, P = 0.004) for the same analysis.The number of pass articles did not correlate with SCI Impact Factors for the top 20 journals  are shown in Table New England Journal of Medicine, JAMA, and Lancet). These three journals and the Cochrane Database of Systematic Reviews provided 56.5% of the articles abstracted, with 28 additional journals providing the other 43.5%. Fifteen titles provided only one article each and, overall, 32 journals provided at least one article for abstraction. Another 51 journals provided at least one article in the "Other Articles Noted" section. Thus, 83 journals from our list of 170 published studies important to internal medicine.The journals contributing articles important to the practice of internal medicine (ACP J Club), the range of NNR for internal medicine was from 40.4 for the Cochrane Database of Systematic Reviews to 1334 for Neurology. For the less stringent definition (article abstracted or noted in ACP J Club), the NNR range for internal medicine was from 3.4 for the Cochrane Database of Systematic Reviews to 242 for Acta Obstetrica et Gynaecologica Scandinavica.The NNR to obtain one high-quality and clinically relevant study or review varied considerably across the titles. For the more stringent definition of clinical relevance . These findings support the findings by Lee et al. [Correlating the number of articles published in e et al.  that SCIEBM) were published in BMJ, Lancet, Cochrane Database of Systematic Reviews, Archives of Disease in Childhood, and Annals of Internal Medicine–these journals provided 55.6% of EBM content . This shows substantial agreement between SCI Impact Factors and number of articles but slightly less agreement than that found using the general internal medicine data (correlation coefficients > 0.688).Correlation analysis showed that the number of qualified articles in each journal title was associated with the journal's SCI Impact Factor . Thirty-two other journals provided articles for abstraction and 33 journals provided studies that were listed only in the "Other Articles Noted" section; 72 journals in total provided content for general care nursing. The NNR for general practice nursing was variable, ranging from 6.0 for Qualitative Health Research to 1530 for New England Journal of Medicine for the more stringent definition of clinical relevance. For the less stringent definition of clinical relevance the NNRs ranged from 4.7 for Qualitative Health Research to 923 for American Journal of Gastroenterology. The low NNR for Qualitative Health Research undoubtedly reflects the fact that only clinical criteria for relevance were applied in the selection of qualitative studies, not explicit methodologic criteria. The reason for lack of methodologic criteria for qualitative studies was that we have been unable to obtain agreement from qualitative researchers of what the quality criteria should be.Nursing content came from many journals, including journals that are considered to be primarily targeted at physicians, and was not concentrated in a small set of journal titles Table . To reacP = 0.57) or the less strict definition (correlation coefficient 0.256, P = 0.038).No correlation was seen between number of articles published per journal title and SCI Impact Factors for either the stringent definition of clinical relevance , with CMAJ having the highest NNR (1007) of those journals with at least one article on mental health. EBMH has a smaller "Other Articles Noted" section. Only eight additional journals provide articles for this section beyond the 61 that provide articles for abstraction.Mental health content was also spread over a broader range of journals than was internal medicine Table . To reacP = 0.02 for the more stringent definition; and correlation coefficient 0.381, P = 0.01 for the less stringent definition).A weak association was shown between the number of published mental health articles and SCI Impact Factors .Combining the content across the four discipline areas, we again see the concentration of important clinically relevant articles in a small subset of journals. Eight journals provided at least one article for abstraction to all four abstract journals: We found that the majority of articles for each discipline were sequestered in a small subset of journals. This is consistent with Bradford's Law of Scattering for journal subsets, which states that the important articles on any topic will be concentrated in a small subset of journals with exponential drop-off in numbers of relevant articles across journal titles . Across Not surprisingly, for broad-based disciplines such as mental health and nursing, the number of titles was greater than for more focused disciplines such as internal medicine. SCI Impact Factors were highly correlated with the number of important clinical articles in separate titles for internal medicine and, to a lesser extent, for general/family practice, and mental health but not for general practice nursing. This likely reflects the volume of clinically important research activity in these fields–with especially high volumes in the disorders managed by internal medicine and its subspecialties–coupled with the avidity of authors from all disciplines to submit their best studies to the high-circulation general journals.et al. [JAMA, Lancet, BMJ, New England Journal of Medicine, and Cochrane Database of Systematic Reviews. Health professionals in all disciplines should be aware that major advances in any field will most likely be published in the main general medicine journals, while at the same time recognizing that specialty journals also publish important information. Much variation exists across journal titles in both the number and proportion of articles that are high quality, clinically important, and newsworthy. Variation also exists across disciplines. It is also interesting to note that all lists of important journals discussed in this report and also the one by Ebell et al. [As found by Weiner et al.  and othel et al.  include EBM, 23 were on the list provided by Ebell et al. (POEM articles) [et al. found common POEMs in 49 journals and any POEMs in 64 journals. POEMs and EBM cover the content of general/family practice by considering a similar number of journals, although both groups read approximately 50% unique titles. Ebell et al. read 85 titles for POEMs articles and we read 170 titles for this study. Our coverage of clinical content was broader and included internal medicine, general practice nursing, and mental health, but 53 titles were read by both groups. Correlational analysis for the ranking of each journal title according to the number of articles identified as clinically important showed a small but significant agreement (0.4397, P = 0.005) when comparing our list with the list by Ebell et al.Of the 45 titles that provided articles to rticles) . Ebell eet al. [Consistent with the data from Weiner et al. , many adJournal Watch. Use of these summaries of studies and reviews can be supplemented by access to fulltext articles.Clinicians in the target disciplines described here could use our findings to focus their fulltext readings. For other disciplines, a similar audit of clinical yield would be needed, either from an appropriate secondary journal that systematically reviews specified journals, or an independent audit. Another approach to staying current may be to subscribe to one or more secondary journals that highlight important clinical advances. These secondary publications have not only selected the most appropriate studies for clinical consideration, they highlight important aspects of methodology and implementation. This assessment of studies before application can be time-consuming and difficult for many clinicians, and involves a certain amount of training and practice to become proficient. Many examples of secondary publications exist in various disciplines and include the four studied in this report, POEMs , and Jouet al. [Many academic medical centers and hospitals provide good online access to major healthcare journals. For example, the Health Sciences Library of the University of Pittsburgh, PA, USA, provides online access to 24 of the top 25 journals in this study and all 25 of the journals identified as high yielders by Ebell et al. . SpecialACP Journal Club ACP J Club AHCPR Agency for Health Care Policy and Research (now AHRQ)AHRQ Agency for Healthcare Research and Quality (formerly AHCPR)CCOHTA Canada Coordinating Office for Health Technology AssessmentEvidence-Based Medicine EBM Evidence-Based Mental Health EBMH Evidence-Based Nursing EBN NNR Number of articles needed to read to obtain one high-quality and clinically relevant study or reviewPOEM Patient-oriented evidence that mattersSCI Science Citation IndexACP Journal Club, Evidence-Based Medicine, Evidence-Based Nursing, and Evidence-Based Mental Health at the time of this study, and were paid for this work, but the publishers of these journals were not involved in the study, which was funded externally. The authors do not hold stocks or shares in any company that may benefit from the publication of this paper.The authors all worked with NLW and RBH prepared grant submissions in relation to this project. All authors drafted and commented on the manuscript and approved the final manuscript, as well as supplied intellectual content to the collection and analysis of the data. NLW and KAM did data collection and analysis, and supervised research staff.The pre-publication history for this paper can be accessed here:Additional File 1 includes a list of the 170 journals read for 2000 along with the number of articles reviewed, the number and percentage that passed criteria, and the NNR . The file name is "Publishing Important Articles Appendix.doc" and it is in Word 2000 format.Click here for file


In sub-Saharan Africa the highest overlap between malaria and HIV infections occurs in female adolescents. Yet control activities for these infections are directed to different target groups, using disparate channels. This reflects the lack of priority given to adolescents and the absence of an accepted framework for delivering health and health-related interventions to this high-risk group. In this paper it is argued that female adolescents require a continuum of care for malaria and HIV – prior to conception, during and after pregnancy and that this should be provided through adolescent services. The evidence for this conclusion is presented. A number of African countries are commencing to formulate and implement adolescent-friendly policies and services and disease control programs for malaria and HIV will need to locate their interventions within such programs to ensure widespread coverage of this important target group. Failure to prioritize adolescent health in this way will seriously limit the success of disease control programs for malaria and HIV prevention. Plasmodium falciparum malaria [The current generation of adolescents – over one billion – is the largest in history  and, far malaria . Biologi malaria . A case  malaria . NeverthPrevalence estimates for HIV infection  for 35 sP. falciparum infection after childhood is also in young, mostly adolescent, women. Data from Malawi . ANC presents an opportunity for HIV prevention, support for discordant couples and care and referral for HIV-infected adolescents. Early assessment is important as the HIV positive individual needs to plan for anti-retroviral treatment (ART) at delivery to prevent mother-to-child transmission. It is also important for malaria control in pregnancy. Intermittent preventive antimalarial treatment (IPT) is the key malaria prevention policy that improves birthweight outcomes and reduces pregnancy anaemia . Its effA major challenge for malaria control is to improve adolescent health prior to the first pregnancy. For non-pregnant adolescents there are several conundrums. On account of drug resistance, malaria combination therapy with artemisinin derivatives is being introduced into an increasing number of sub-Saharan African countries. Artemisinin may be foeto-toxic and should be avoided, except for symptomatic infections with multi-drug resistant malaria, in the first trimester of pregnancy . InadverA further issue is that the benefits of IPT extend to pregnant but not nulliparous adolescents who may be anaemic or parasitaemic at the start of their pregnancy. The use of permethrin-treated bednets would protect against anaemia and parasitaemia during the first trimester, but adolescent pregnant girls and their newborns are the least likely to be net users .The growing literature on adolescent-friendly health services shows that adolescents have substantial concerns about the delivery of interventions through health services . Almost The only country in Africa that has scaled up adolescent health services is South Africa . The SouWhat about young men? Available evidence suggests that young men are not attracted to clinic services which are perceived as "female "spaces . In GhanBehavioural change, including appropriate health-seeking practices, is essential for disease reduction. A decline in HIV prevalence amongst adolescents has been reported from Zambia , Uganda Few developing countries have well-defined policies for delivering care to the non-pregnant or pregnant adolescent, especially for sexual health. Existing malaria guidelines also do not mention adolescents specifically .An important aspect is the issue of the adolescent's right to sexual and reproductive information and services vis à vis the legitimate rights of parents to act in the best interests of a child, and the health care provider's right to work within the law. These are issues of consent and confidentiality for adolescents considered "minors" and under parental control, or wives under their husband's or husband's family's jurisdiction. When these issues are ignored, adolescents cannot be assured of confidentiality, and health workers are neither bound, nor protected by, the legal system. Rights established in law, or by signed Conventions (eg Convention of the Rights of the Child), may not be implemented. Health policy falls down because it is not explicit about the vulnerability of female adolescents and does not challenge cultural and social norms that can perpetuate the disease burden. Similarly, encouragement for improving female literacy and education may be lacking, reducing the supportive environment for promoting uptake of adolescent health services and safer sex. Advocacy is essential to co-ordinate law, custom and health practice. At another level, it is important to acknowledge that interventions for female adolescents raise ethical issues that are avoided when they are excluded from care. The policy of avoiding treatment of adolescent girls (and adult women) because they might be in the first trimester of pregnancy, raises issues of gender discrimination and has been a serious issue for parasitic disease control programs . ReducinFor both HIV and malaria, although interventions are available to reduce the disease burden, adolescents are not prioritized. Effective policies and services are needed to reduce the substantial disease burden they experience from these infections. To secure this goal, concerted action across HIV and malaria disease control programs is required. This will reinforce support for adolescent programs rather than weakening them by multiple separate activities, which can confuse implementation. A schedule of activities is required that is reinforced by other systems (e.g. education) to facilitate embedding it into a way of life. These issues need to be addressed head-on by the adolescent health community to achieve the focus needed, and to establish the realistic steps forward. In the short term, developing services to deliver HIV and malaria interventions can act as a catalyst for addressing broader adolescent health service needs. In the medium-term adolescent-friendly health services provide the required platform for the evaluation and delivery of future vaccines and drug therapies for HIV, malaria  and otheBoth authors made substantial contributions to the analysis, interpretation, drafting and revision of this article. Both have agreed to authorship.


KAR2, cannot account for the transcriptional regulation of most genes in this set. To address this puzzle, we analyzed the promoters of UPR target genes computationally, identifying as candidate UASs short sequences that are statistically overrepresented. We tested the most promising of these candidate UASs for biological activity, and identified two novel UPREs, which are necessary and sufficient for UPR activation of promoters. A genetic screen for activators of the novel motifs revealed that the transcription factor Gcn4p plays an essential and previously unrecognized role in the UPR: Gcn4p and its activator Gcn2p are required for induction of a majority of UPR target genes during ER stress. Both Hac1p and Gcn4p bind target gene promoters to stimulate transcriptional induction. Regulation of Gcn4p levels in response to changing physiological conditions may function as an additional means to modulate the UPR. The discovery of a role for Gcn4p in the yeast UPR reveals an additional level of complexity and demonstrates a surprising conservation of the signaling circuit between yeast and metazoan cells.Eukaryotic cells respond to accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), a signal transduction pathway that communicates between the ER and the nucleus. In yeast, a large set of UPR target genes has been experimentally determined, but the previously characterized unfolded protein response element (UPRE), an upstream activating sequence (UAS) found in the promoter of the UPR target gene  The yeast unfolded protein response activates a large set of target genes, but a characterized element found in the promoter of one target, KAR2, cannot account for most targets. Using computational and experimental methods, the authors identify additional elements, as well a role for GCN4p in the response The vast majority of all cellular secretory and membrane proteins are folded and modified in the endoplasmic reticulum (ER), from which they are transported to their final destination in the secretory pathway. When the protein folding capacity of the ER is exceeded or experimentally impaired, unfolded proteins accumulate in the ER and activate the unfolded protein response (UPR). The UPR allows the ER to communicate with the nucleus , where aS. cerevisiae, unfolded ER proteins stimulate the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1p  . The UPRire1 or Δhac1 cells.The set of 381 genes was defined by microarray hybridization expression profiling, using a stringent quantitative “filter” that required the expression profile of each target gene to closely match that of previously known and well-characterized UPR target genes. In particular, the filter demanded that the expression profile of a target gene closely correlate to that of canonical UPR targets over a time course of UPR induction, and that induction be significantly greater in wild-type (WT) than in either ΔKAR2/BiP promoter , which is modeled as having been composed by concatenating “words” (short oligonucleotides) drawn from a probabilistic “dictionary” according to their frequencies. To infer the dictionary from the observed text, we employed the previously developed computational algorithm, MobyDick, which was developed based on a probabilistic segmentation model , 2000b. We first constructed a dictionary from the UPR target gene promoters. To this end, we compiled a text from the promoters of all 381 UPR target genes as previously defined . We defiBecause words with similar sequences are likely to possess similar biological activity, we considered groups of related words as units in our subsequent analysis. We grouped the dictionary into motifs by performing every possible pairwise alignment between all words, and then clustering words with high mutual alignment scores. A motif may contain two or more words, or just a single word. For a multiword motif, the words defining the motif are similar to one another and share common core sequences . The cluP of this many or more appearances occurring by chance. A small P value (high −log10P) indicates that the motif is overrepresented relative to the expectation. 10P for each motif plotted against this rank. We chose the eight highest-ranking motifs (open circles) as candidates for experimental testing (We reasoned that motifs that are likeliest to represent bona fide regulatory elements will be nonrandomly distributed in the genome and appear more often in the UPR target gene promoters than expected by chance. Therefore, we counted the number of times each motif (i.e., a sequence match to any of the words the motif comprises) appeared in the approximately 6,000 promoters in the genome, and computed from this figure the frequency with which each motif would be expected to appear in a promoter if it were distributed randomly throughout the genome. We then counted the number of times the motif was actually found in the 381 target promoters and calculated the probability  testing B, analyzlacZ reporter construct that contains a crippled version of the CYC1 promoter that is transcriptionally silent in the absence of a UAS  A, or ditlacZ reporter constructs derived from two native promoters in which the motifs appear. We chose for UPRE-2 the promoter of ERO1, encoding an ER resident redox protein, and for UPRE-3 the promoter of DHH1, encoding an RNA helicase. Both genes are robust targets of the UPR . Similarly, WT but not Δire1 or Δhac1 cells bearing the UPRE-3-containing DHH1-promoter-driven reporter expressed higher levels of β-galactosidase after treatment with DTT (DHH1 promoter entirely eliminated induction by ER stress ( “− UPRE-3” columns).To test whether these motifs are also necessary for transcriptional activation, we designed  the UPR  and lackTaken together, the data presented so far indicate that, as with the classical UPRE-1, UPRE-2 and UPRE-3 are both sufficient A and neccis-regulatory elements that differ in sequence from the canonical UPRE-1 suggests that trans-activating factors other than Hac1p may bind these elements. Alternatively, Hac1p, alone or accompanied by another factor or factors, may be able to recognize multiple sequences. To distinguish between these possibilities and potentially reveal novel regulatory factors, we attempted to identify genes which, when overexpressed, activate transcription of the UPRE-2 reporter plasmid in the absence of an ER stress signal. The design of this screen recapitulates the approach which identified HAC1 as a high-copy activator of the UPRE-1  genomic DNA library  colonies were recovered and rescreened by the same assay. Plasmids from positively rescreened clones were retransformed into the Δire1 UPRE-2-lacZ strain to verify plasmid linkage of the activator phenotype.We transformed a strain bearing the UPRE-2- library . A Δire1IRE1 locus and surrounding sequences; IRE1 has been previously shown to be activated by overexpression and is a high-copy activator of UPRE-1  shared the f UPRE-1 . RecoverGCN4 locus. GCN4 encodes a bZIP transcription factor, which has been well-characterized as a component of the cellular response to amino acid starvation and other stresses , indicating that overexpression of GCN4 is sufficient to stimulate transcription from the UPRE-2-driven reporter gene in the absence of ER stress, Ire1p activity, or Hac1p production. We also starved cells for histidine by administering 3-aminotriazole (3-AT), which induces translation of Gcn4p (GCN4, amino acid starved cells exhibited a significant increase of UPRE-2 transcription in the absence of ER stress ( “+3-AT, −Tm” columns). GCN4 overexpression alone did not activate transcription from either UPRE-1 or UPRE-3 reporter genes, emphasizing that these motifs are not synonymous with UPRE-2.The second class of positive inserts (eight plasmids) shared the stresses  but has activity A. GCN4 oof Gcn4p . As whenGCN4 overexpression is sufficient to activate transcription from a UPRE-2 reporter, we next asked whether GCN4 is also necessary to activate transcription in response to ER stress. We deleted GCN4 from strains bearing UPRE-1, UPRE-2, and UPRE-3 reporter constructs and assayed β-galactosidase activity in response to UPR activation. Upon UPR induction, HAC1 mRNA was spliced normally, and Hac1p was produced at WT levels in Δgcn4 mutants (unpublished data). However, Δgcn4 cells failed to induce transcription, not only of the UPRE-2-driven reporter but also of the UPRE-1- and UPRE-3-driven reporters , suggesting that GCN4 activity is limiting for UPR-dependent transcription from all three UPREs. Similarly, stimulation of Gcn4p production by amino acid starvation also increases the magnitude of the transcriptional response ; when eIF-2α is phosphorylated, scanning ribosomes fail to initiate at upstream ORFs encoded by the GCN4 5′ UTR and are able to initiate translation at the GCN4 ORF itself , and the set of genes previously identified as UPR-independent (“nontargets”) . The disire1 cells (a and b, e and f, i and j, and m and n). This is the case both for target genes bearing any UPRE in the promoter (a–l) as well as the remainder of the target set for which a UPRE has not been identified (m–p). For those genes with an identified UPRE in their promoters, expression patterns in both Δgcn4 (c, g, and k) and Δgcn2 mutants (d, h, and l) show trends similar to those in Δire1. In both mutants, the sets of genes whose promoters contain a UPRE are significantly less upregulated relative to their induction in the WT. Some UPR target genes exhibit residual upregulation in Δgcn4 and Δgcn2, suggesting that these promoters have only a partial requirement for GCN4/GCN2. This effect is most prominent for genes containing UPRE-1 in the Δgcn2 mutant (j), where the residual induction crosses the threshold into marginal statistical significance ; it is possible that the residual levels of Gcn4p present in a Δgcn2 mutant are sufficient to allow UPR transcription from these promoters, or alternatively that UPRE-1 promoters are relatively less sensitive to Gcn4p levels (and concomitantly, relatively more reliant on Hac1p) for induction (see Discussion). In contrast, induction of the “no UPRE” genes is quite high in Δgcn4 and Δgcn2 cells (o and p versus m). As a population, these genes are not significantly less upregulated in the mutants than in the WT. It would appear that the UPREs identified to date define a special subset of UPR target genes that are responsive not only to IRE1 and HAC1 but that are particularly sensitive to the GCN4/GCN2 branch of the pathway.The majority of the genes in the nontarget set , as previously reported (In the context of other stress responses (e.g., amino acid starvation), Gcn4p levels are regulated via phosphorylation of eIF-2α by Gcn2p . Becausereported . We obsereported B.GCN4 by ER stress mediated by increased phospho-eIF-2α. Here, we observe increased Gcn4p levels under ER stress conditions even when phospho-eIF-2α levels are not detectably altered.These data are consistent with two possibilities: first, that Gcn2p is responsible for both basal levels of Gcn4p and its induction upon ER stress; or second, that Gcn2p is responsible only for maintaining basal levels of Gcn4p, while another pathway mediated by Ire1p/Hac1p further elevates Gcn4p levels during the UPR. If Gcn2p is responsible for upregulation of Gcn4p during the UPR, we should observe a concomitant increase in the level of eIF-2α phosphorylation. We did not observe such an increase . It is pGCN4 plays an essential role in the UPR, with a knockout phenotype closely resembling that of Δire1 and Δhac1: the absence of any of these genes prevents transcriptional activation by ER stress. This observation could be a consequence of one of several different mechanisms: Gcn4p might act upstream or downstream of Hac1p in the same linear pathway, or act in a parallel pathway that converges at target promoters. Two lines of evidence from data already introduced argue that Gcn4p does not act upstream of Hac1p. First, GCN4 overexpression is sufficient to activate transcription from UPRE-2 in a Δhac1 mutant (see hac1 mutant (see tant see A, indicatant see A, indicagcn4 mutant (HAC1 gene lacking the intron is sufficient to activate transcription from the UPRE-1 (ire1, +Hac1p” columns). In the absence of GCN4, however, the constitutive expression of Hac1p does not activate transcription from any of the three reporter constructs (gcn4, +Hac1p” columns), suggesting that Hac1p's function at promoters containing any one of the three UPREs requires the presence of Gcn4p. Thus, Gcn4p must act at the same point as or downstream of Hac1p. Following the same line of reasoning, for UPRE-1 and UPRE-3, GCN4 overexpression alone is insufficient to activate transcription in the absence of HAC1 (e.g., see hac1 mutants), indicating that at UPRE-containing promoters Hac1p must act at the same point as or downstream of Gcn4p. Thus, the observations enumerated here are consistent with the interpretation that Gcn4p and Hac1p act together at target gene promoters.Further evidence that Gcn4p does not act upstream of Hac1p is provided by the observation that expression of Hac1p cannot activate transcription in a Δ4 mutant . In a WTKAR2 promoter (oligo 1), used in previous experiments demonstrating direct binding of Hac1p to UPRE-1 (ERO1 promoter (oligo 2). 32P-labeled oligonucleotides were incubated with cell extracts and subjected to native (nondenaturing) polyacrylamide gel electrophoresis, and visualized by autoradiography (To explore this possibility directly, we performed gel-retardation assays with the UPRE-1-containing segment of the o UPRE-1 , and theiography .ire1 mutant (lanes 9 and 12), in which Hac1p cannot be synthesized. Likewise, in both Δgcn4 and Δgcn2 mutants, the binding activity observed in WT cells was absent. In both Δgcn4 and Δgcn2 mutants, however, a faster migrating complex appeared, which likely represents Hac1p alone binding the oligos (lanes 10, 11, 13, and 14).As previously observed, oligo 1's mobility was retarded when incubated with crude extracts from UPR-induced cells, but not extracts from untreated cells . Although the two new UPRE sequences look at first glance entirely different from the well-characterized UPRE-1, one of them may share “half-site” similarity: UPRE-2 has a three base identity with UPRE-1 at the 3′ end (TACGTG versus CAGNGTG); whether these bases make equivalent contacts with the bound transcription factors remains to be determined. Taken together, the sequence diversity of the motifs conferring similar transcriptional control upon binding of the same transcriptional activators illustrates the difficulty of predicting biological regulation from promoter sequences alone, even if binding sites in one context are well defined experimentally.Beginning only with the set of genes induced by the UPR and the promoter sequences of all genes in the genome, we computationally identified candidate motifs that obeyed the statistical properties we would expect of regulatory sequences, i.e., high frequency in UPR target promoters, and enrichment in the target promoters relative to the rest of the promoters in the genome. Two of these motifs, UPRE-2 and UPRE-3, are both necessary and sufficient to confer ER stress responsiveness in an cis-acting elements known to act in the UPR.The identification of these novel sequences allows a greater proportion of UPR target gene regulation to be explained within the paradigm of modular transcriptional control, i.e., in which a “portable” sequence module (a UAS) located within a promoter confers pathway responsiveness on the gene in question. The two novel motifs described triple the number of target genes whose regulation can be described in terms of a modular control mechanism, thus adding significantly to the repertoire of And yet, the resulting description of UPR transcription remains incomplete, as approximately 50% of the target genes still lack a recognizable UPREs. It may be that more biologically active motifs exist among the 109 motifs that emerged from the overrepresentation analysis, as many of the untested motifs are overrepresented relative to chance in the UPR target set by many orders of magnitude. For the eight motifs tested, we tested whether a motif was necessary for promoter induction only if it had already been shown to be sufficient in the artificial promoter system. Because of this experimental approach, it remains possible that some motifs not found to be sufficient are dependent for their activity on some contextual parameter (e.g., particular nearby flanking sequences). Thus it may be that some UPREs are not generally portable to other contexts, but are nonetheless necessary for UPR responsiveness of the native promoters in which they reside. Also, particularly rare motifs would have been omitted from the dictionary; thus, it is possible that complementary computational approaches might allow detection of uncommon motifs that this analysis missed. Finally, some UASs may remain ultimately undiscoverable within the paradigm of modular regulation. Motifs that are particularly sensitive to chromatin structure or position relative to the transcription initiation site would not be detected by an approach that neglected these parameters.ERO1 after removal of UPRE-2 .A plausible resolution to this paradox may be that the UPRE-1 is heavily dependent on context. The experiments that defined the key core nucleotides proceeded by single point mutation at each position while holding constant the identity of all other nucleotides from the source 22-bp stretch of the  species A. This cDespite these qualifications, the approach has successfully uncovered novel information about how the UPR is regulated. The appealing aspect of the strategy described here is that such studies are not limited to the UPR but can be generally employed in the study of any transcriptional response in any organism for which promoter sequences for all genes are known and in which the comprehensive genomic output of the response can be measured by expression profiling. The sole requirement of the probabilistic segmentation/overrepresentation computations is that a partition of the genome (into “target genes” and “nontarget genes”) be made on the basis of some meaningful difference in expression levels under the conditions of interest; the analysis thereafter proceeds by comparing the distribution of candidate motifs in the target gene set and the remainder of the genome. Further refinement of the mathematical tools therefore promises to be of invaluable help in our quest for a comprehensive understanding of the logic and complex interactions of transcriptional programs in eukaryotic cells.The overexpression screen for activators of UPRE-2 revealed a role for the transcription factor Gcn4p, which we show to be required not only for activity of UPRE-2 but for all three known UPREs. Gcn4p and its upstream activator Gcn2p thus join Ire1p, Hac1p, and Rlg1p in the list of essential players in the yeast UPR.GCN4 encodes a well-characterized transcription factor acting in several distinct stress responses including amino acid starvation, glucose limitation, and ultraviolet irradiation , which retains some expression of Gcn4p. In the absence of Hac1p but in the presence of high concentrations of Gcn4p (e.g., when GCN4 is overexpressed), Gcn4p can bind the UPRE-2 on its own, either as a homodimer or a monomer.The basis for this differential affinity for Gcn4p is strongly suggested by a refined consensus sequence for UPRE-2, and is illustrated by multiple species alignment of the promoter B. We seapromoter . The res compare A and 7B The transient upregulation of Gcn4p levels, which we observe upon UPR induction, may therefore serve to increase the transcriptional output of the response, especially early in the response. Most UPR target genes are robustly induced after 15 min of ER stress ; hence, GCN4 for normal UPR induction are those that are most urgently required by the cell under specific conditions, under which UPR is induced and Gcn4p levels are high for reasons unrelated to ER stress. The relationship between the cellular stress responses that regulate Gcn4p and the potentiation of UPR transcription will therefore be an important subject for future study.Gcn4p itself mediates a broad transcriptional program in response to a diverse set of cellular conditions and stresses . The recIRE1 and HAC1 mediate the transient increase in Gcn4p remains to be elucidated. Given that Hac1p and Gcn4p are observed in the same complex with DNA, one intriguing possibility is that association with Hac1p serves to stabilize Gcn4p.The mechanism by which HAC1 mRNA splicing retains its role as the “switch” that turns the UPR on or off. Gcn4p, whose levels appear to be limiting for the extent of gene regulation, would therefore play a role in setting the “gain” or “volume” of the response, perhaps allowing communication from other stress response pathways in the cell. Such a gain control could serve as an adjunct to the “Super-UPR” (S-UPR) gain control described in the accompanying paper  and of a mechanism for transcriptional regulation of Hac1p . MobyDick generated a dictionary of 328 words. We filtered out words that were too short, appeared in too many copies (such as AT-rich short repeats), or were of low quality . With the filters number_of_copies < 200, length > 4, and quality_factor > 0.2, we obtained 201 words.To build the dictionary of putative regulatory elements for UPR target genes, we first extracted the 600-bp upstream regions of all UPR target genes. To get rid of simple repeats unlikely to be regulatory elements (such as AT-rich repeats and transposable elements), we removed exact repeats of lengths 15 bp or longer, and kept the remaining fragments of lengths longer than 50 bp. What remained was the input sequence for the dictionary construction. We used the MobyDick algorithm based on probabilistic segmentation  to buildx mutates to any other given base y with probability p/3, and remains the same base with a probability (1 − p). The score for a pair x–y is given by the log-odds-ratio of observing the pair under the mutation model versus observing the pair at random. With the choice of p = 0.5 , a matching pair scores ln(2), and a mismatch scores ln(2/3). We normalized the scores to fall between 0 and 1 by the largest score. We then used the CAST algorithm to group words into clusters, with the threshold parameter set at 0.7 . This procedure generated 109 motifs.Using the filtered dictionary, we grouped similar words into motifs using the clustering algorithm CAST , as follexpN in the UPR target gene promoters based on the genome-wide frequencies. We then counted the observed number of occurrences obsN of the motif in the promoters of UPR target genes. We used Poisson statistics to calculate the probability P of observing a number of occurrences equal to or greater than obsN by chance, based on expN. The test based on Poisson statistics is a very good approximation of the more rigorous test based on the binomial distribution, where the probability P is the probability of seeing a specific instance of the motif in the UPR gene set and the total number of trials tN is the total number of copies of the motif in the genome. Since P is small (0.059) and tN is large (ranging from approximately ten to approximately 1000) but the product expN is finite, the resulting distribution is well approximated by a Poisson distribution with mean = expN.To test which motifs are significantly overrepresented in the promoters of UPR target genes, we counted for each motif the total number of occurrences in all promoters, and calculated the expected number of occurrences S. cerevisiae, S. bayanus, S. mikatae, S. paradoxus, and S. kudriavevii  UPRE. Intact promoter reporter constructs (pPW668–pPW671) used in ERO1 or DHH1 initiation codon, or by two fragments spanning the same sequence but with the UPRE motif replaced by a restriction site. The high-copy GCN4 plasmid (pPW672) used in GCN4 ORF. Source sequence contexts, olignucleotide sequences, and select PCR primers are compiled in HAC1 used in HAC1 (pPW353/pJC316) used in UPRE reporter constructs used in B, and 5  the UPR  and, if GCN4 and GCN2 and the integrated GCN4-myc were constructed by PCR cassette/generic primer mutagenesis  prior to addition of drug.HIS3 gene; for histidine-deprived cultures, overnight cultures were washed three times in SD-histidine, then diluted to low density in SD-histidine and grown to midlog phase before the addition of the drug.DTT (Sigma, St. Louis, Missouri, United States) was added to cultures to a final concentration of 2 mM. Tm (Boehringer Mannheim, Indianapolis, Indiana, United States) was added to cultures to a final concentration of 1 μg/ml. 3-AT (Sigma) was added to cultures to a final concentration of 10 mM. All 3-AT treatments were performed on strains WT for the To assay β-galactosidase activity on solid growth media, we overlaid plates with buffered soft agar containing X-gal (Sigma) as described previously . For liqStrains were grown in YPD (pH 5.4) as in http://derisilab.ucsf.edu/more) and was performed as described previously  using a z-score scheme. For a given gene set and a given strain, we calculated the average fold induction for genes in the set and compared it to the value for the genome overall. The null hypothesis was that the selected gene set was no different from a randomly selected set  from the genome overall. Under this hypothesis, the average μ has a distribution well approximated by a normal distribution  with mean genomeμ and standard deviation σ/√(Nset), where setN is the total number of genes in the test set. We computed a z-score, z=√(Nset)(μ−μgenome)/σ, which should have a standard normal distribution (zero mean and unit variance) under the null hypothesis. The P value was calculated by integrating the standard normal curve from z to infinity.We tested the statistical significance of the induction for the three gene sets (UPRE-1, UPRE-2, and UPRE-3 genes) in four different strains . For sequences, see Gel shifts were performed as previously described  except tTable S1N, the average number of times the string is delimited as a word among all segmentations of the data; Xi, the number of matches of the word anywhere in the text; p, the frequency of drawing the word from the dictionary, optimized over all words to give the maximum likelihood of observing the text; Z = p + sp, where sp is the probability with which the word can be made by combining shorter words from the dictionary; sig = significance = Np/sqrt(N[Z − p]).This table contains an alphabetical list of the dictionary “words” compiled by the MobyDick algorithm from the “text” comprising the promoters of UPRE target genes. Associated statistics for each word are as follows: (10 KB TXT).Click here for additional data file.Table S2tot is the number of times a given motif appeared in the promoters of the genome overall; Nexp is the number of times one would expect a given motif to appear in the 381 promoters of UPR target genes if the motif were distributed randomly throughout all promoters; Nobs is the number of times a given motif actually appears in the target gene promoters; and −log10P is a measure of overrepresentation based on Poisson statistics (P is the likelihood that a given observed distribution would occur by chance).N(3 KB TXT).Click here for additional data file.Table S3KAR2 and ERO1 promoters, derived from S. cerevisiae and related budding yeasts. Asterisk indicates 100% conserved residues. Scer, S. cerevisiae; Skud, S. kudriavevii; Spar, S. paradoxus; Smik, S. mikatae; Sbay, S. bayanus.This table contains CLUSTALW alignments for the (8 KB TXT).Click here for additional data file.Table S4This table contains the sequences of primers and olignonucleotide sequences used in construction of plasmids for this study, as well as oligonucleotide sequences used for probes in the gel-shift analysis.(38 KB DOC).Click here for additional data file.The GenBank accession numbers of the gene products discussed in this paper are Dhh1p (NP_010121), Ero1p (NP_013576), Gcn4p (NP_010907), Gcn2p (NP_010569), Hac1p (NP_011946), and Ire1p (NP_116622).Microarray data can be accessed at the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) database as platform number GPL1001 and sample numbers GSM16985–GSM1988.


Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery. Resin-linked oligonucleotides are described that efficiently partition subpools of DNA based on sequence, enabling DNA- programmed chemical synthesis The information is amplifiable, so that a single molecule can be copied to produce a measurable quantity of nucleic acid. A large collection of enzymatic tools (e.g., polymerases, helicases, recombinases, and restriction enzymes) and man-made tools  exist to manipulate DNA. Several technologies take advantage of these facts. For example, patterned DNA fragments have been used to direct self-assembly of nucleic acid objects , second position (a2, b2, and so on), and subsequent positions. Codons present in one coding position are mutually exclusive of the codons at any other position. The identity of the first codon determines the fate of the gene at the first decision point of the network, the second codon at the second decision point, and so forth.Our experimental scheme is illustrated in Genes are “read” by hybridization to a set of anticodon columns. Each anticodon column displays an oligonucleotide complementary to one codon sequence, and a complete set of columns comprises the complements to all codons at a single coding position. Genes bind to the columns by codon–anticodon base pairing, and are thus partitioned. To read a subsequent coding position, the genes are first transferred to a nonspecific DNA binding resin, regenerating an unhy-bridized state. This DNA is then hybridized to a new set of anticodon columns. By a series of such reading cycles, the sequence of a gene guides it through the network.8 unique genes contained routing instructions for eight decision points.For DNA routing genes, we adopted a modular design adaptable to networks of varying depth and width. We chose codons consisting of 20 bases, catenated to neighboring codons through 20-base noncoding regions . To prev1, a2, . . . , a8) into ten 340-base genes . The ten different genes were subcloned. Eight 60-base segments were then PCR amplified from an equimolar mixture of the parental plasmids. Each segment consisted of a coding position and two adjacent noncoding regions. The eight degenerate products were spliced together into 340-base fragments by primerless PCR, thus producing a library of 108 complexity. In principle, collective assembly of the 160 40mer oligonucleotides would have created the library in one step. In practice, the two-stage approach provided better control over codon distributions in the final gene population.Construction of the gene library proceeded in two stages A. InitiaThe noncoding regions play an instrumental role in the construction and handling of genes. By providing conserved crossover points, they facilitate the modular generation of highly complex populations from a small number of starting oligonucleotides. For the same reason, the noncoding regions make it possible to diversify existing gene sets by recombination. The noncoding regions also place codons in the correct coding position, ensuring that all genes incorporate one codon per branch point of a network. The existence of a well-defined “reading frame” results from the fact that anticodon columns only hybridize to DNA subsequences at a specified coding position, and not to codons elsewhere in the gene.To obtain hybridization-capable nucleic acid, the duplex DNA genes must be converted to a single-stranded form. (Possibly, duplex DNA hybridization to oligonucleotides through D-loops could be driven by RecA and ATP ). A numbSynthesis of anticodon columns involves covalent attachment of oligonucleotides to a solid phase. Thiol-containing and amine-containing oligonucleotide modification reagents are commercially available, and either should facilitate coupling to an appropriately activated resin. However, pilot experiments indicated that amide linkages were more easily prepared than thioether linkages. The deprotection protocols for oligonucleotide-linked sulfhydryl moieties were more complex than for amine moieties, and sulfhydryl-modified oligonucleotides were prone to oxidation and general loss during manipulation steps. Amine-modified oligonucleotides were easier to work with and were thus used for production of anticodon columns. It proved necessary to desalt crude oligonucleotides over reverse-phase cartridges before coupling.N-hydroxysuccinimide[NHS]-activated Sepharose, carbonyl diimidazole-activated Sephacryl S-1000). 20-base modified anticodon oligonucleotides were coupled to the resins. Quantification by reverse-phase chromatography of the uncoupled oligonucleotide remaining in solution provided a measure of reaction progress , methacrylate , and agarose  analysis of the load, flow-through, and elute fractions . By thisFollowing an observation that long polyethylene glycol (PEG) linkers dramatically improve hybridization to DNA on polypropylene surfaces , we syntWe next investigated how buffer conditions and temperature influenced the accuracy and yield of 340-base ssDNA hybridization to anticodon columns. For these experiments, a single radiolabeled DNA gene was diluted 10-fold into an excess (50 pmol) of an unlabeled routing gene library, and loaded onto a 250-μl diethylaminoethyl (DEAE) Sepharose column. The DEAE Sepharose column was placed in a closed 3-ml fluid circuit containing ten anticodon columns, of which only one complemented a codon within the radiolabeled DNA. Hybridization buffer was pumped over the system in a direction that placed the complementary anticodon column distal to the DEAE Sepharose column , left. ABy varying the temperature (25 °C to 70 °C), salt identity (sodium chloride, lithium chloride, or tetramethylammonium chloride) and salt concentration (10 mM to 2 M) of the hybridization buffer, we determined that 1.5 M sodium chloride in a phosphate buffered solution with pH 6.5 at 45 °C provided the most robust hybridization behavior over multiple codon sequences. In addition, an initial high temperature step (70 °C) and the presence of a DEAE column inline proved critical to achieving uniformly high yields . The higIn order to route a DNA fragment through successive levels of a hierarchical tree, multiple hybridization steps are required. DNA from parental anticodon columns must be isolated and hybridized to anticodon columns corresponding to the daughter-node branches. The manipulations must be highly efficient to ensure good routing yields through trees with many levels.n for n hybridizations.We investigated several schemes for accomplishing iterative hybridizations. Our initial strategies utilized a multi-step procedure with three columns (anticodon, DEAE, and reverse-phase), linear transfer formats, and centrifugal evaporation. These three-column strategies did not prove to be high-yielding. We eventually observed that efficient iterative hybridizations could be accomplished with only two columns, using cyclic column-to-column transfers. Thus, a parental anticodon column with bound DNA was placed in a liquid circuit with a 250-μl DEAE-Sepharose column , right. The final requirement was to isolate DNA as a concentrated, salt-free aqueous solution upon completion of routing. For this purpose, DNA bound to anticodon columns was eluted with a small volume of an (ethylenedinitrilo)tetraacetic acid (EDTA) solution and precipitated. Alternatively, DNA bound to DEAE-Sepharose columns was transferred to a reverse-phase cartridge by cyclically pumping a high-salt buffer over the two columns in series. DNA on the reverse-phase cartridge was then washed with deionized water, eluted in acetonitrile/water, concentrated by evaporation, and desalted over a microcentrifuge gel-filtration column.Escherichia coli SSB or RecA  polyethylene glycol of average molecular weight 1500 Da  and all other chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). DEAE columns were prepared by pipetting approximately 250 μl of DEAE Sepharose Fast Flow resin  into empty TWIST column housings .1–a8), b-type codons (b1–b8), and so forth. Each set of 16 oligonucleotides was assembled into a 340-base fragment by primerless PCR , 500 μM EDTA, and 500 μM EGTA. Acrylamide was removed by spin column filtration, and the solution volume was reduced to 800 μl by centrifugal evaporation. Samples were phenol/chlorofom extracted, ethanol precipitated, and resuspended in water.ssDNA was generated using a modified NASBA reaction . Duplex The crude Jeffamine material was purified by fast protein liquid chromatography cation-exchange chromatography over a 5-ml Hi-Trap SP column (Amersham Biosciences #17-1152-01). In early work, 1-ml batches of a 250-mg/ml aqueous solution were loaded onto the column in 50 mM acetic acid, washed with load buffer, and bumped off with 1 M lithium chloride. Subsequently, we developed a gradient protocol. The material was loaded in water, and the product was eluted with a linear water–hydrogen-chloride gradient (0–30 mM hydrogen chlo-ride over 15 column volumes) at 6 °C monitored by refractive index detection . After every fifth injection, the column was washed with 1.5 M sodium chloride to remove a yellow residue and was then reequilibrated in deionized water. Pooled fractions of the bisamine peak were brought to pH 10 by addition of solid sodium carbonate, and the purified Jeffamine product (compound 1) was extracted into methylene chloride. The combined organic layers were dried over sodium sulfate, and solvent was removed by rotary evaporation. Yields of the pale yellow solid were 40% based on the weight of crude starting material.One mole equivalent of 1.5 M glutaric anhydride in dioxane was added to a briskly stirred 250-mg/ml aqueous solution of purified Jeffamine (compound 1). After 30 min, the crude reaction product was injected in 1-ml batches over a 5-ml Hi-Trap SP column and eluted as described above. Pooled fractions of the monoamine peak were brought to pH 7 by addition of solid sodium bicarbonate, and the purified linker product (compound 2) was extracted into methylene chloride, dried over sodium sulfate, and obtained as a pale yellow solid by rotary evaporation. Yields were 35% to 50% based on the weight of purified Jeffamine starting material. A compound similar to the purified linker compound 2 is commercially available .To prepare resin compound 3, compound 1 or compound 2 was dissolved at 300 mg/ml in DMF/200 mM DIEA. The linker solution was incubated with one volume equivalent of drained NHS-activated Sepharose (Amersham Biosciences #17-0906-01). The suspension was rotated at 37 °C for 72 h, washed over a plastic frit with DMF to remove excess linker, and incubated with 1 M ethanolamine in DMF for an additional 12 h at 37 °C. The product resin was washed and stored at 6 °C. Resins coupled to compound 1 were further treated by incubation with an equal volume of DMF containing 100 mM glutaric anhydride and 15 mM pyridine at 37 °C under rotation for 48 h.Typically, TBTU (320 mg) and NHS (115 mg) were dissolved in 4 ml of DMF/500 mM DIEA, and drained compound 3 (1 ml) was added. The suspension was rotated at 37 °C for 1 h. Product resin was washed with the following sequence (20 ml of each): ethyl acetate, tetrahydrofuran, ethanol, water, 5 M sodium chloride, water, and DMF. Resin activation was performed just prior to oligonucleotide coupling.Twenty-base capture oligonucleotides were synthesized using standard phosphoramidite chemistry, with the addition of a C12-methoxytritylamine modifier at the 5′-end (Glen Research #10-1912). Following ammonia cleavage and drying, the oligonucleotides were desalted over C18 Sep-Pak cartridges . Purification proceeded according to the manufacturer's instructions, but a deionized water wash was inserted before the final elution step to remove residual TEAA. Coupling reactions of oligonucleotides to resin were carried out in low-binding 0.65-ml microcentrifuge tubes . Ten nanomoles of a capture oligonucleotide and 10 nmol of a nonaminated 10mer control oligonucleotide in a 40-μl aqueous solution were mixed with 160 μl of DMF/200 mM DIEA. Fifty microliters of drained resin compound 4 was added, and the suspension was rotated at 37 °C for 12 h. Reaction progress was monitored by HPLC. Supernatant aliquots (20 μl) were injected onto a 4.6-mm × 25-cm Varian Microsorb-MV 300-5 C18 column  and eluted with a linear water–acetonitrile gradient (0%–45% acetonitrile in five column volumes) in the presence of 0.1 M TEAA (pH 5.2) at 50 °C. After 12 h, the resin was pelleted by centrifugation at 100 g for 1 min, supernatant was removed, and the resin was incubated with 1 M ethanolamine in DMF for an additional 12 h at 37 °C. The derivatized resins were loaded into empty DNA synthesis column housings .Hybridization was performed in a closed system consisting of an anticodon column, male tapered luer couplers (Biosearch Technologies #CL-1504-1), capillary tubing (Amersham Biosciences #19-7477-01), silicone tubing , tubing connectors (Amersham Biosciences #19-2150-01, #18-1003-68, and #18-1027-62), and a peristaltic pump (Amersham Biosciences #18-1110-91). Approximately 1 ml of hybridization buffer (60 mM sodium phosphate (pH 6.5), 1.5 M sodium chloride, 10 mM EDTA, and 0.005% Triton X-100) containing 400 pmol of a complementary 20-base oligonucleotide and 400 pmol of a noncomplementary ten-base oligonucleotide was cyclically pumped through the system at 0.5 ml/min for 1 h in a 46-°C water bath. DNA was eluted off the anticodon column with 4 ml of 1 mM EDTA (pH 8.0) and 0.005% Triton X-100 heated to 80 °C. Flow-through and elute fractions were analyzed by HPLC as described above (0%–18% acetonitrile in five column volumes).35S preset channel of a scintillation counter .ssDNA was loaded onto a DEAE-Sepharose column as described . AnticodDEAE and anticodon columns were connected in series using male tapered luer couplers, 3.16-mm manifold tubing , and tygon tubing 3603. Using a peristaltic pump , approximately 7 ml of a 50% DMF solution was flowed cyclically over the columns at 3 ml/min for either 1 h at 45 °C or 12 h at 25 °C.DEAE columns were connected in series to C8 SepPak columns (Waters #WAT036775) using male tapered luer couplers, tygon tubing 3603, and tubing connectors. Approximately 6 ml of 50 mM ethanolamine (pH 10.0), 1.5 M sodium chloride, 1 mM EDTA, and 0.005% Triton X-100 was cyclically pumped over the columns at 1 ml/min for 1 h at 50 °C. The Sep-Pak columns were then washed with 12 ml of 100 mM TEAA (pH 6.5) followed by 12 ml of water. ssDNA was eluted from the Sep-Pak column with 4 ml of 50% acetonitrile heated to 80 °C. Samples were concentrated by centrifugal evaporation to a volume of approximately 30 μl and desalted over G25 Sephadex spin columns (Sigma-Aldrich #G-25-150).