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Background/Objective: SeptiCyte RAPID is a transcriptional host response assay that discriminates between sepsis and non-infectious systemic inflammation (SIRS) with a one-hour turnaround time. The overall performance of this test in a cohort of 419 patients has recently been described [Balk et al., J Clin Med 2024, 13, 1194]. In this study, we present the results from a detailed stratification analysis in which SeptiCyte RAPID performance was evaluated in the same cohort across patient groups and subgroups encompassing different demographics, comorbidities and disease, sources and types of pathogens, interventional treatments, and clinically defined phenotypes. The aims were to identify variables that might affect the ability of SeptiCyte RAPID to discriminate between sepsis and SIRS and to determine if any patient subgroups appeared to present a diagnostic challenge for the test. Methods: (1) Subgroup analysis, with subgroups defined by individual demographic or clinical variables, using conventional statistical comparison tests. (2) Principal component analysis and k-means clustering analysis to investigate phenotypic subgroups defined by unique combinations of demographic and clinical variables. Results: No significant differences in SeptiCyte RAPID performance were observed between most groups and subgroups. One notable exception involved an enhanced SeptiCyte RAPID performance for a phenotypic subgroup defined by a combination of clinical variables suggesting a septic shock response. Conclusions: We conclude that for this patient cohort, SeptiCyte RAPID performance was largely unaffected by key variables associated with heterogeneity in patients suspected of sepsis.

(1) Background: SeptiCyte RAPID is a molecular test for discriminating sepsis from non-infectious systemic inflammation, and for estimating sepsis probabilities. The objective of this study was the clinical validation of SeptiCyte RAPID, based on testing retrospectively banked and prospectively collected patient samples. (2) Methods: The cartridge-based SeptiCyte RAPID test accepts a PAXgene blood RNA sample and provides sample-to-answer processing in ~1 h. The test output (SeptiScore, range 0-15) falls into four interpretation bands, with higher scores indicating higher probabilities of sepsis. Retrospective (N = 356) and prospective (N = 63) samples were tested from adult patients in ICU who either had the systemic inflammatory response syndrome (SIRS), or were suspected of having/diagnosed with sepsis. Patients were clinically evaluated by a panel of three expert physicians blinded to the SeptiCyte test results. Results were interpreted under either the Sepsis-2 or Sepsis-3 framework. (3) Results: Under the Sepsis-2 framework, SeptiCyte RAPID performance for the combined retrospective and prospective cohorts had Areas Under the ROC Curve (AUCs) ranging from 0.82 to 0.85, a negative predictive value of 0.91 (sensitivity 0.94) for SeptiScore Band 1 (score range 0.1-5.0; lowest risk of sepsis), and a positive predictive value of 0.81 (specificity 0.90) for SeptiScore Band 4 (score range 7.4-15; highest risk of sepsis). Performance estimates for the prospective cohort ranged from AUC 0.86-0.95. For physician-adjudicated sepsis cases that were blood culture (+) or blood, urine culture (+)(+), 43/48 (90%) of SeptiCyte scores fell in Bands 3 or 4. In multivariable analysis with up to 14 additional clinical variables, SeptiScore was the most important variable for sepsis diagnosis. A comparable performance was obtained for the majority of patients reanalyzed under the Sepsis-3 definition, although a subgroup of 16 patients was identified that was called septic under Sepsis-2 but not under Sepsis-3. (4) Conclusions: This study validates SeptiCyte RAPID for estimating sepsis probability, under both the Sepsis-2 and Sepsis-3 frameworks, for hospitalized patients on their first day of ICU admission.

SeptiCyte ® RAPID is a gene expression assay measuring the relative expression levels of host response genes PLA2G7 and PLAC8, indicative of a dysregulated immune response during sepsis. As severe forms of COVID-19 may be considered viral sepsis, we evaluated SeptiCyte RAPID in a series of 94 patients admitted to Foch Hospital (Suresnes, France) with proven SARS-CoV-2 infection. EDTA blood was collected in the emergency department (ED) in 67 cases, in the intensive care unit (ICU) in 23 cases and in conventional units in 4 cases. SeptiScore (0-15 scale) increased with COVID-19 severity. Patients in ICU had the highest SeptiScores, producing values comparable to 8 patients with culture-confirmed bacterial sepsis. Receiver operating characteristic (ROC) curve analysis had an area under the curve (AUC) of 0.81 for discriminating patients requiring ICU admission from patients who were immediately discharged or from patients requiring hospitalization in conventional units. SeptiScores increased with the extent of the lung injury. For 68 patients, a chest computed tomography (CT) scan was performed within 24 h of COVID-19 diagnosis. SeptiScore >7 suggested lung injury ≥50% (AUC = 0.86). SeptiCyte RAPID was compared to other biomarkers for discriminating Critical + Severe COVID-19 in ICU, versus Moderate + Mild COVID-19 not in ICU. The mean AUC for SeptiCyte RAPID was superior to that of any individual biomarker or combination thereof. In contrast to C-reactive protein (CRP), correlation of SeptiScore with lung injury was not impacted by treatment with anti-inflammatory agents. SeptiCyte RAPID can be a useful tool to identify patients with severe forms of COVID-19 in ED, as well as during follow-up.

Tools for the evaluation of COVID-19 severity would help clinicians with triage decisions, especially the decision whether to admit to ICU. The aim of this study was to evaluate SeptiCyte RAPID, a host immune response assay (Immunexpress, Seattle USA) as a triaging tool for COVID-19 patients requiring hospitalization and potentially ICU care. SeptiCyte RAPID employs a host gene expression signature consisting of the ratio of expression levels of two immune related mRNAs, PLA2G7 and PLAC8, measured from whole blood samples. Blood samples from 146 adult SARS-CoV-2 (+) patients were collected within 48 h of hospital admission in PAXgene blood RNA tubes at Hospital del Mar, Barcelona, Spain, between July 28th and December 1st, 2020. Data on demographics, vital signs, clinical chemistry parameters, radiology, interventions, and SeptiCyte RAPID were collected and analyzed with bioinformatics methods. The performance of SeptiCyte RAPID for COVID-19 severity assessment and ICU admission was evaluated, relative to the comparator of retrospective clinical assessment by the Hospital del Mar clinical care team. In conclusion, SeptiCyte RAPID was able to stratify COVID-19 cases according to clinical severity: critical vs. mild (AUC = 0.93, p < 0.0001), critical vs. moderate (AUC = 0.77, p = 0.002), severe vs. mild (AUC = 0.85, p = 0.0003), severe vs. moderate (AUC = 0.63, p = 0.05). This discrimination was significantly better (by AUC or p-value) than could be achieved by CRP, lactate, creatine, IL-6, or D-dimer. Some of the critical or severe cases had "early" blood draws (before ICU admission; n = 33). For these cases, when compared to moderate and mild cases not in ICU (n = 37), SeptiCyte RAPID had AUC = 0.78 (p = 0.00012). In conclusion, SeptiCyte RAPID was able to stratify COVID-19 cases according to clinical severity as defined by the WHO COVID-19 Clinical Management Living Guidance of January 25th, 2021. Measurements taken early (before a patient is considered for ICU admission) suggest that high SeptiScores could aid in predicting the need for later ICU admission., (© 2023. The Author(s).)

Background: There is an urgent need to develop biomarkers that stratify risk of bacterial infection in order to support antimicrobial stewardship in emergency hospital admissions., Methods: We used computational machine learning to derive a rule-out blood transcriptomic signature of bacterial infection (SeptiCyte™ TRIAGE) from eight published case-control studies. We then validated this signature by itself in independent case-control data from more than 1500 samples in total, and in combination with our previously published signature for viral infections (SeptiCyte™ VIRUS) using pooled data from a further 1088 samples. Finally, we tested the performance of these signatures in a prospective observational cohort of emergency department (ED) patients with fever, and we used the combined SeptiCyte™ signature in a mixture modelling approach to estimate the prevalence of bacterial and viral infections in febrile ED patients without microbiological diagnoses., Results: The combination of SeptiCyte™ TRIAGE with our published signature for viral infections (SeptiCyte™ VIRUS) discriminated bacterial and viral infections in febrile ED patients, with a receiver operating characteristic area under the curve of 0.95 (95% confidence interval 0.90-1), compared to 0.79 (0.68-0.91) for WCC and 0.73 (0.61-0.86) for CRP. At pre-test probabilities 0.35 and 0.72, the combined SeptiCyte™ score achieved a negative predictive value for bacterial infection of 0.97 (0.90-0.99) and 0.86 (0.64-0.96), compared to 0.90 (0.80-0.94) and 0.66 (0.48-0.79) for WCC and 0.88 (0.69-0.95) and 0.60 (0.31-0.72) for CRP. In a mixture modelling approach, the combined SeptiCyte™ score estimated that 24% of febrile ED cases receiving antibacterials without a microbiological diagnosis were due to viral infections. Our analysis also suggested that a proportion of patients with bacterial infection recovered without antibacterials., Conclusions: Blood transcriptional biomarkers offer exciting opportunities to support precision antibacterial prescribing in ED and improve diagnostic classification of patients without microbiologically confirmed infections.

Objectives: Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is a leading cause of death and disability among children worldwide. Identifying sepsis in pediatric patients is difficult and can lead to treatment delay. Our objective was to assess the host proteomic response to infection utilizing an aptamer-based multiplexed proteomics approach to identify novel serum protein changes that might help distinguish between pediatric sepsis and infection-negative systemic inflammation and hence can potentially improve sensitivity and specificity of the diagnosis of sepsis over current clinical criteria approaches., Design: Retrospective, observational cohort study., Setting: PICU and cardiac ICU, Seattle Children's Hospital, Seattle, WA., Patients: A cohort of 40 children with clinically overt sepsis and 30 children immediately postcardiopulmonary bypass surgery (infection-negative systemic inflammation control subjects) was recruited. Children with sepsis had a confirmed or suspected infection, two or more systemic inflammatory response syndrome criteria, and at least cardiovascular and/or pulmonary organ dysfunction., Interventions: None., Measurements and Main Results: Serum samples from 35 of the sepsis and 28 of the bypass surgery subjects were available for screening with an aptamer-based proteomic platform that measures 1,305 proteins to search for large-scale serum protein expression pattern changes in sepsis. A total of 111 proteins were significantly differentially expressed between the sepsis and control groups, using the linear models for microarray data (linear modeling) and Boruta (decision trees) R packages, with 55 being previously identified in sepsis patients. Weighted gene correlation network analysis helped identify 76 proteins that correlated highly with clinical sepsis traits, 27 of which had not been previously reported in sepsis., Conclusions: The serum protein changes identified with the aptamer-based multiplexed proteomics approach used in this study can be useful to distinguish between sepsis and noninfectious systemic inflammation.

Background: The performance of a new diagnostic test is typically evaluated against a comparator which is assumed to correspond closely to some true state of interest. Judgments about the new test's performance are based on the differences between the outputs of the test and comparator. It is commonly assumed that a small amount of uncertainty in the comparator's classifications will negligibly affect the measured performance of a diagnostic test., Methods: Simulated datasets were generated to represent typical diagnostic scenarios. Comparator noise was introduced in the form of random misclassifications, and the effect on the apparent performance of the diagnostic test was determined. An actual dataset from a clinical trial on a new diagnostic test for sepsis was also analyzed., Results: We demonstrate that as little as 5% misclassification of patients by the comparator can be enough to statistically invalidate performance estimates such as sensitivity, specificity and area under the receiver operating characteristic curve, if this uncertainty is not measured and taken into account. This distortion effect is found to increase non-linearly with comparator uncertainty, under some common diagnostic scenarios. For clinical populations exhibiting high degrees of classification uncertainty, failure to measure and account for this effect will introduce significant risks of drawing false conclusions. The effect of classification uncertainty is magnified further for high performing tests that would otherwise reach near-perfection in diagnostic evaluation trials. A requirement of very high diagnostic performance for clinical adoption, such as a 99% sensitivity, can be rendered nearly unachievable even for a perfect test, if the comparator diagnosis contains even small amounts of uncertainty. This paper and an accompanying online simulation tool demonstrate the effect of classification uncertainty on the apparent performance of tests across a range of typical diagnostic scenarios. Both simulated and real datasets are used to show the degradation of apparent test performance as comparator uncertainty increases., Conclusions: Overall, a 5% or greater misclassification rate by the comparator can lead to significant underestimation of true test performance. An online simulation tool allows researchers to explore this effect using their own trial parameters (https://imperfect-gold-standard.shinyapps.io/classification-noise/) and the source code is freely available (https://github.com/ksny/Imperfect-Gold-Standard)., Competing Interests: Leo C. McHugh and Thomas D. Yager declare that they are present or past employees and shareholders of Immunexpress. Leo C. McHugh declares that he is an inventor on the following patents and patent applications filed by Immunexpress: PCT/AU2017/050894 (“Systemic Inflammatory and Pathogen Biomarkers and Uses Therefor”), PCT/AU2016/050927 (“Pathogen Biomarkers and Uses Therefor”), PCT/AU2016/051269 (“Triage Biomarkers and Uses Therefor”), US 15/160,749 (“Validating Biomarker Measurement”), PCT/AU2016/250388 (“Validating Biomarker Measurement”), US Patent 10,167,511 (“Biomarker Identification”), US Patent 10,190,169 (“Biomarker Identification”), US 14/714,188 (“Biomarker signature method, and apparatus and kits therefor”), US 14/616,565 (“Biomarker signature method, and apparatus and kits therefor”), PCT/AU2015/050043 (“Biomarker signature method, and apparatus and kits therefor”). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Differentiating sepsis from the systemic inflammatory response syndrome (SIRS) in critical care patients is challenging, especially before serious organ damage is evident, and with variable clinical presentations of patients and variable training and experience of attending physicians. Our objective was to describe and quantify physician agreement in diagnosing SIRS or sepsis in critical care patients as a function of available clinical information, infection site, and hospital setting., Methods: We conducted a post hoc analysis of previously collected data from a prospective, observational trial ( N  = 249 subjects) in intensive care units at seven US hospitals, in which physicians at different stages of patient care were asked to make diagnostic calls of either SIRS, sepsis, or indeterminate, based on varying amounts of available clinical information (clinicaltrials.gov identifier: NCT02127502). The overall percent agreement and the free-marginal, inter-observer agreement statistic kappa ( κ free ) were used to quantify agreement between evaluators (attending physicians, site investigators, external expert panelists). Logistic regression and machine learning techniques were used to search for significant variables that could explain heterogeneity within the indeterminate and SIRS patient subgroups., Results: Free-marginal kappa decreased between the initial impression of the attending physician and (1) the initial impression of the site investigator ( κ free 0.68), (2) the consensus discharge diagnosis of the site investigators ( κ free 0.62), and (3) the consensus diagnosis of the external expert panel ( κ free 0.58). In contrast, agreement was greatest between the consensus discharge impression of site investigators and the consensus diagnosis of the external expert panel ( κ free 0.79). When stratified by infection site, κ free for agreement between initial and later diagnoses had a mean value + 0.24 (range - 0.29 to + 0.39) for respiratory infections, compared to + 0.70 (range + 0.42 to + 0.88) for abdominal + urinary + other infections. Bioinformatics analysis failed to clearly resolve the indeterminate diagnoses and also failed to explain why 60% of SIRS patients were treated with antibiotics., Conclusions: Considerable uncertainty surrounds the differential clinical diagnosis of sepsis vs. SIRS, especially before organ damage has become highly evident, and for patients presenting with respiratory clinical signs. Our findings underscore the need to provide physicians with accurate, timely diagnostic information in evaluating possible sepsis., Competing Interests: Ethics approval was gained from the relevant institutional review boards: Intermountain Medical Center/Latter Day Saints Hospital (1024931); Johns Hopkins Hospital (IRB00087839); Rush University Medical Center (15111104-IRB01); Loyola University Medical Center (208291); Northwell Healthcare (16-02-42-03); and Grady Memorial Hospital (000-87806).This manuscript does not contain any individual person’s data in any form. Therefore, consent for publication is not required.The authors have read the journal’s policy and declare the following competing interests: LM, TDY, AR, RBB, RAB, and TS are current or past employees and/or shareholders of Immunexpress.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rationale: A molecular test to distinguish between sepsis and systemic inflammation of noninfectious etiology could potentially have clinical utility., Objectives: This study evaluated the diagnostic performance of a molecular host response assay (SeptiCyte LAB) designed to distinguish between sepsis and noninfectious systemic inflammation in critically ill adults., Methods: The study employed a prospective, observational, noninterventional design and recruited a heterogeneous cohort of adult critical care patients from seven sites in the United States (n = 249). An additional group of 198 patients, recruited in the large MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium trial in the Netherlands ( www.clinicaltrials.gov identifier NCT01905033), was also tested and analyzed, making a grand total of 447 patients in our study. The performance of SeptiCyte LAB was compared with retrospective physician diagnosis by a panel of three experts., Measurements and Main Results: In receiver operating characteristic curve analysis, SeptiCyte LAB had an estimated area under the curve of 0.82-0.89 for discriminating sepsis from noninfectious systemic inflammation. The relative likelihood of sepsis versus noninfectious systemic inflammation was found to increase with increasing test score (range, 0-10). In a forward logistic regression analysis, the diagnostic performance of the assay was improved only marginally when used in combination with other clinical and laboratory variables, including procalcitonin. The performance of the assay was not significantly affected by demographic variables, including age, sex, or race/ethnicity., Conclusions: SeptiCyte LAB appears to be a promising diagnostic tool to complement physician assessment of infection likelihood in critically ill adult patients with systemic inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT01905033 and NCT02127502).

The innate immune system of humans and other mammals responds to pathogen-associated molecular patterns (PAMPs) that are conserved across broad classes of infectious agents such as bacteria and viruses. We hypothesized that a blood-based transcriptional signature could be discovered indicating a host systemic response to viral infection. Previous work identified host transcriptional signatures to individual viruses including influenza, respiratory syncytial virus and dengue, but the generality of these signatures across all viral infection types has not been established. Based on 44 publicly available datasets and two clinical studies of our own design, we discovered and validated a four-gene expression signature in whole blood, indicative of a general host systemic response to many types of viral infection. The signature's genes are: Interferon Stimulated Gene 15 (ISG15), Interleukin 16 (IL16), 2',5'-Oligoadenylate Synthetase Like (OASL), and Adhesion G Protein Coupled Receptor E5 (ADGRE5). In each of 13 validation datasets encompassing human, macaque, chimpanzee, pig, mouse, rat and all seven Baltimore virus classification groups, the signature provides statistically significant (p < 0.05) discrimination between viral and non-viral conditions. The signature may have clinical utility for differentiating host systemic inflammation (SI) due to viral versus bacterial or non-infectious causes.

Objectives: SeptiCyte Lab (Immunexpress, Seattle, WA), a molecular signature measuring the relative expression levels of four host messenger RNAs, was developed to discriminate critically ill adults with infection-positive versus infection-negative systemic inflammation. The objective was to assess the performance of Septicyte Lab in critically ill pediatric patients., Design: Prospective observational study., Setting: Pediatric and Cardiac ICUs, Seattle Children's Hospital, Seattle, WA., Patients: A cohort of 40 children with clinically overt severe sepsis syndrome and 30 children immediately postcardiopulmonary bypass surgery was recruited. The clinically overt severe sepsis syndrome children had confirmed or highly suspected infection (microbial culture orders, antimicrobial prescription), two or more systemic inflammatory response syndrome criteria (including temperature and leukocyte criteria), and at least cardiovascular ± pulmonary organ dysfunction., Interventions: None (observational study only)., Measurements and Main Results: Next-generation RNA sequencing was conducted on PAXgene blood RNA samples, successfully for 35 of 40 (87.5%) of the clinically overt severe sepsis syndrome patients and 29 of 30 (96.7%) of the postcardiopulmonary bypass patients. Forty patient samples (~ 60% of cohort) were reanalyzed by reverse transcription-quantitative polymerase chain reaction, to check for concordance with next-generation sequencing results. Postcardiopulmonary bypass versus clinically overt severe sepsis syndrome descriptors included the following: age, 7.3 ± 5.5 versus 9.0 ± 6.6 years; gender, 41% versus 49% male; Pediatric Risk of Mortality, version III, 7.0 ± 4.6 versus 8.7 ± 6.4; Pediatric Logistic Organ Dysfunction, version II, 5.1 ± 2.2 versus 4.8 ± 2.8. SeptiCyte Lab strongly differentiated postcardiopulmonary bypass and clinically overt severe sepsis syndrome patients by receiver operating characteristic curve analysis, with an area-under-curve value of 0.99 (95% CI, 0.96-1.00). Equivalent performance was found using reverse transcription-quantitative polymerase chain reaction. There was no significant correlation between the score produced by the SeptiCyte Lab test and measures of illness severity, immune compromise, or microbial culture status., Conclusions: SeptiCyte Lab is able to discriminate clearly between clinically well-defined and homogeneous postcardiopulmonary bypass and clinically overt severe sepsis syndrome groups in children. A broader investigation among children with more heterogeneous inflammation-associated diagnoses and care settings is warranted.

Background: Systemic inflammation is a whole body reaction having an infection-positive (i.e., sepsis) or infection-negative origin. It is important to distinguish between these two etiologies early and accurately because this has significant therapeutic implications for critically ill patients. We hypothesized that a molecular classifier based on peripheral blood RNAs could be discovered that would (1) determine which patients with systemic inflammation had sepsis, (2) be robust across independent patient cohorts, (3) be insensitive to disease severity, and (4) provide diagnostic utility. The goal of this study was to identify and validate such a molecular classifier., Methods and Findings: We conducted an observational, non-interventional study of adult patients recruited from tertiary intensive care units (ICUs). Biomarker discovery utilized an Australian cohort (n = 105) consisting of 74 cases (sepsis patients) and 31 controls (post-surgical patients with infection-negative systemic inflammation) recruited at five tertiary care settings in Brisbane, Australia, from June 3, 2008, to December 22, 2011. A four-gene classifier combining CEACAM4, LAMP1, PLA2G7, and PLAC8 RNA biomarkers was identified. This classifier, designated SeptiCyte Lab, was validated using reverse transcription quantitative PCR and receiver operating characteristic (ROC) curve analysis in five cohorts (n = 345) from the Netherlands. Patients for validation were selected from the Molecular Diagnosis and Risk Stratification of Sepsis study (ClinicalTrials.gov, NCT01905033), which recruited ICU patients from the Academic Medical Center in Amsterdam and the University Medical Center Utrecht. Patients recruited from November 30, 2012, to August 5, 2013, were eligible for inclusion in the present study. Validation cohort 1 (n = 59) consisted entirely of unambiguous cases and controls; SeptiCyte Lab gave an area under curve (AUC) of 0.95 (95% CI 0.91-1.00) in this cohort. ROC curve analysis of an independent, more heterogeneous group of patients (validation cohorts 2-5; 249 patients after excluding 37 patients with an infection likelihood of "possible") gave an AUC of 0.89 (95% CI 0.85-0.93). Disease severity, as measured by Sequential Organ Failure Assessment (SOFA) score or Acute Physiology and Chronic Health Evaluation (APACHE) IV score, was not a significant confounding variable. The diagnostic utility of SeptiCyte Lab was evaluated by comparison to various clinical and laboratory parameters available to a clinician within 24 h of ICU admission. SeptiCyte Lab was significantly better at differentiating cases from controls than all tested parameters, both singly and in various logistic combinations, and more than halved the diagnostic error rate compared to procalcitonin in all tested cohorts and cohort combinations. Limitations of this study relate to (1) cohort compositions that do not perfectly reflect the composition of the intended use population, (2) potential biases that could be introduced as a result of the current lack of a gold standard for diagnosing sepsis, and (3) lack of a complete, unbiased comparison to C-reactive protein., Conclusions: SeptiCyte Lab is a rapid molecular assay that may be clinically useful in managing ICU patients with systemic inflammation. Further study in population-based cohorts is needed to validate this assay for clinical use.

Objective: This study was designed to test the utility of a blood-based approach to identify mild osteoarthritis (OA) of the knee., Methods: Blood samples were drawn from 161 subjects, including 85 subjects with arthroscopically diagnosed mild OA of the knee and 76 controls. Following RNA isolation, an in-house custom cDNA microarray was used to screen for differentially expressed genes. A subset of selected genes was then tested using real-time RT-PCR. Logistic regression analysis was used to evaluate linear combinations of the biomarkers and receiver operating characteristic curve analysis was used to assess the discriminatory power of the combinations., Results: Genes differentially expressed (3543 genes) between mild knee OA and control samples were identified through microarray analysis. Subsequent real-time RT-PCR verification identified six genes significantly down-regulated in mild OA: heat shock 90kDa protein 1, alpha; inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein; interleukin 13 receptor, alpha 1; laminin, gamma 1; platelet factor 4 (also known as chemokine (C-X-C motif) ligand 4) and tumor necrosis factor, alpha-induced protein 6. Logistic regression analysis identified linear combinations of nine genes--the above six genes, early growth response 1; alpha glucosidase II alpha subunit; and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)--as discriminatory between subjects with mild OA and controls, with a sensitivity of 86% and specificity of 83% in a training set of 78 samples. The optimal biomarker combinations were then evaluated using a blind test set (67 subjects) which showed 72% sensitivity and 66% specificity., Conclusions: Linear combinations of blood RNA biomarkers offer a substantial improvement over currently available diagnostic tools for mild OA. Blood-derived RNA biomarkers may be of significant clinical value for the diagnosis of early, asymptomatic OA of the knee.

We present the first comprehensive transcriptome-to-genome mapping for human cartilage. First, we determined that the cartilage transcriptome represents between 13,200 and 15,800 unique genes. Next, a subset of approximately 10,000 of the best characterized cartilage-expressed transcripts (CETs) was selected and mapped to the human genome. The distribution of CETs across the genome was found to be significantly different compared to the expected distribution. Furthermore, clusters of adjacent coordinately transcribed genes, as well as numerous "hot spots" and "cold spots" for transcription in cartilage, were identified. We propose that transcriptional control in cartilage can be exerted over genomic domains containing as few as four neighboring genes. Our findings, which are consistent with recent "chromatin domain" models of transcription, are further supported by our identification of CETs that putatively encode components of the HDAC- and Swi/SNF-mediated chromatin remodeling pathways. Our study illustrates the value of comprehensive high-resolution scans to detect transcription patterns within the human genome.

We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.

We present a new method for the linearization and alignment of data traces generated by multilane automated DNA sequencing instruments. Application of this method to data generated with the Visible Genetics Open Gene DNA sequencing system (using MicroCel 700 gel cassettes, with a 25 cm separation distance) allows read lengths of > 1,000 nucleotides to be routinely obtained with high confidence and > 97% accuracy. This represents an increase of 10-15% in average read length, relative to data from this system that have not been processed in the fashion described herein. Most importantly, the linearization and alignment method allows usable sequence to be obtained from a fraction of 10-15% of data sets which, because of original trace misalignment problems, would otherwise have to be discarded. Our method involves adding electrophoretic calibration standards to the DNA sequencing fragments. The calibration standards are labeled with a dye that differs spectrally from the dye attached to the sequencing fragments. The calibration standards are identical in all the lanes. Analysis of the mobilities of the calibration standards allows correction for both systematic and random variation of electrophoretic properties between gel lanes. We have successfully used this method with two-dye and three-dye DNA sequencing instruments.

The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 microm thick polyacrylamide gels. Two DNA ladders can be analyzed simultaneously in each of 16 lanes on a gel, after labeling with far-red absorbing dyes such as Cy5 and Cy5.5. This allows a simultaneous bidirectional sequencing of four templates. We have evaluated the Clipper sequencer, by cycle-sequencing of an M13 single-stranded DNA standard, and by coupled amplification and sequencing (CLIP) of reverse-transcribed human immunodeficiency virus (HIV-1) RNA standards and clinical patient samples. (i) Limitations of instrument. We have examined basic instrument parameters such as detector stability, background, digital sampling rate, and gain. With proper usage, the optical and electronic subsystems of the Clipper sequencer do not limit the data collection or sequence-determination processes. (ii) Limitations of gel performance. We have also examined the physics of DNA band separation on 50 microm thick MicroCel gels. We routinely obtain well-resolved sequence which can be base-called with 98.5% accuracy to position approximately 450 on an 11 cm gel, and to position approximately 900 on a 25 cm gel. Resolution on 5 and 11 cm gels ultimately is limited by a sharp decrease in spacing between adjacent bands, in the biased reptation separation regime. Fick's (thermal) diffusion appears to be of minor importance on 6 cm or 11 cm gels, but becomes an additional resolution-limiting factor on 25 cm gels. (iii) Limitations of enzymology. Template quality, primer nesting, choice of DNA polymerase, and choice between dye primers and dye terminators are key determinants of the ability to detect mutations and polymorphisms on the Clipper sequencer, as on other DNA sequencers. When CLIP is used with dye-labeled primers and a DNA polymerase of the F667Y, delta(5'--> 3' exo) class, we can routinely detect single-nucleotide mutations and polymorphisms over the 0.35-0.65 heterozygosity range. We present an example of detecting therapeutically relevant mutations in a clinical HIV-1 RNA isolate.

In this study, we demonstrate the developmental activation, in the zebrafish embryo, of a surveillance mechanism which triggers apoptosis to remove damaged cells. We determine the time course of activation of this mechanism by exposing embryos to camptothecin, an agent which specifically inhibits topoisomerase I within the DNA replication complex and which, as a consequence of this inhibition, also produces strand breaks in the genomic DNA. In response to an early (pre-gastrula) treatment with camptothecin, apoptosis is induced at a time corresponding approximately to mid-gastrula stage in controls. This apoptotic response to a block of DNA replication can also be induced by early (pre-MBT) treatment with the DNA synthesis inhibitors hydroxyurea and aphidicolin. After camptothecin treatment, a high proportion of cells in two of the embryo's three mitotic domains (the enveloping and deep cell layers), but not in the remaining domain (the yolk syncytial layer), undergoes apoptosis in a cell-autonomous fashion. The first step in this response is an arrest of the proliferation of all deep- and enveloping-layer cells. These cells continue to increase in nuclear volume and to synthesize DNA. Eventually they become apoptotic, by a stereotypic pathway which involves cell membrane blebbing, "margination" and fragmentation of nuclei, and cleavage of the genomic DNA to produce a nucleosomal ladder. Fragmentation of nuclei can be blocked by the caspase-1,4,5 inhibitor Ac-YVAD-CHO, but not by the caspase-2,3,7[, 1] inhibitor Ac-DEVD-CHO. This suggests a functional requirement for caspase-4 or caspase-5 in the apoptotic response to camptothecin. Recently, Xenopus has been shown to display a developmental activation of the capability for stress- or damaged-induced apoptosis at early gastrula stage. En masse, our experiments suggest that the apoptotic responses in zebrafish and Xenopus are fundamentally similar. Thus, as for mammals, embryos of the lower vertebrates exhibit the activation of surveillance mechanisms, early in development, to produce the selective apoptosis of damaged cells., (Copyright 1999 Academic Press.)

We describe a method for preparing nuclear spreads from cells of live, unfixed zebrafish embryos at the late-gastrula (approximately 8000 cell) stage of development. The method consists of a sequence of four steps: (1) a slow, gentle lysis, in low to moderate salt concentration, of cells and then nuclei, to release DNA-containing fibres; (2) spreading of the released fibres by a transverse fluid flow; (3) electrostatic, and possibly also covalent, attachment of the spread fibers to poly(L-lysine)-coated glass microscope slides; and (4) continued incubation to produce periodic cleavage of the DNA within the fibres, apparently through activation of endogenous nucleases. The nuclear spreads are imaged with epifluorescence, at a spatial resolution approaching the Rayleigh limit (approximately 230 nm for blue light). The epifluorescent signal is provided from Hoechst 33,258 bound specifically to the DNA, from a dye-coupled antibody conjugate bound specifically to histone H1 in the fibres, or from a DNA nick end-labelling assay. The spontaneous cleavage of DNA-containing fibres in step (4) of the above procedure can be blocked by the chelating agents EGTA and EDTA, by the caspase-2,3,7 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde, and by the caspase-1,4,5 inhibitors N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone. These data suggest that the spontaneous cleavage of fibres is catalysed by nucleases that become activated through a caspase-mediated mechanism. The involvement of caspase-dependent nucleases would suggest that an apoptosis pathway is activated in the spreads during their prolonged incubation. If bona fide apoptosis is induced in living zebrafish embryos by treatment with camptothecin (a topoisomerase I poison), and then nuclear spreads are prepared, we observe a similar fragmentation of the spread fibres. However, in this case the fragmentation is more rapid and complete. We hypothesize that, during the early phase of apoptosis, one or more endogenous nucleases are activated by a caspase-mediated mechanism. The nuclease(s) then specifically recognize and cleave a susceptible, periodically repeating feature of interphase chromatin.

The bone morphogenetic proteins, BMP-2 and OP-1, are candidates for growth factors that control renal branching morphogenesis. We examined their effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting duct morphogenesis (mIMCD-3 cells are derived from the terminal inner medullary collecting duct of the SV40 mouse). Osteogenic protein-1 (OP-1), at a dose of 0.25 nM, increased explant growth by 30% (P = 0.001). In contrast, 100-fold greater concentrations of OP-1 (28 nM) decreased explant growth by 10% (P < 0.001). BMP-2 was entirely inhibitory (maximum inhibition of 7% at 5 nM, P < 0.0004). In an in vitro model for branching morphogenesis utilizing the kidney epithelial cell line, mIMCD-3, low doses of OP-1 (< 0.5 nM) increased the number of tubular structures formed by 28 +/- 5% (P = 0.01), whereas concentrations > 0.5 nM decreased that number by 22 +/- 8% (P = 0.02). All concentrations of BMP-2 (0.05-10 nM) were inhibitory (maximum inhibition at 10 nM of 88 +/- 3%, P < 0.0001). Stimulatory doses of OP-1 increased tubular length (P = 0.003) and the number of branch points/structure (3.2-fold increase, P = 0.0005) compared with BMP-2. To determine the molecular basis for these effects, we demonstrated that BMP-2 is bound to mIMCD-3 cells by the type I serine/threonine kinase receptor, ALK-3, and that OP-1 bound to an approximately 80-kDa protein using ligand-receptor affinity assays. To demonstrate that OP-1 can exert both stimulatory and inhibitory effects within a developing kidney, embryonic explants were treated with agarose beads saturated with 2 microM OP-1. OP-1 decreased the number of ureteric bud/collecting duct branches adjacent to the beads by 58 +/- 1% (P < 0.0001). In contrast, the number of branches in tissue distal to the OP-1 beads was enhanced, suggesting a stimulatory effect at lower doses of OP-1. We conclude that OP-1 and BMP-2 directly control branching morphogenesis and that the effects of OP-1 are dependent on its local concentration within developing kidney tissue.

We have studied the developmental activation of the metaphase checkpoint, and the consequences of activating this checkpoint, in the zebrafish embryo. (1) Treatment with nocodazole (a microtubule destabiliser) before mid-blastula transition (MBT) produces complete destruction of all nuclei in the deep cell layer of the embryo. In contrast, nocodazole treatment after MBT efficiently produces metaphase arrest in this cell layer. Thus, the metaphase checkpoint becomes activated at MBT. (2) Although a metaphase arrest is induced by nocodazole, it is not induced by paclitaxel (a microtubule stabiliser). Thus the metaphase checkpoint appears to sense a destabilisation, but not a stabilisation, of spindle microtubules. (3) Metaphase-arrested cells (in nocodazole) can be driven into the next interphase by adding the Ca2+-specific ionophore A23187. Thus, a Ca2+-signalling pathway lies downstream of, or parallel to, the metaphase checkpoint. (4) After mid-gastrula stage, treatment with nocodazole produces DNA fragmentation in all three cell layers. In the enveloping epithelial monolayer (EVL), this is associated with a classical apoptotic phenotype. In the deep layer, it is associated with an unusual, highly condensed nuclear state that is entered directly from metaphase arrest. Thus, after the mid-gastrula stage, the embryo responds to nocodazle by undergoing apoptosis. (5) Nocodazole-induced apoptosis in the deep cell layer can be blocked by the caspase-1,4,5 inhibitors Ac-YVAD-CHO and Ac-YVAD-CMK. This suggests that a homologue of the C. elegans ced-9-ced-4-ced-3 pathway is involved in control over apoptosis in the early zebrafish embryo.

We address the developmental activation, in the zebrafish embryo, of intrinsic cell-cycle checkpoints which monitor the DNA replication process and progression through the cell cycle. Eukaryotic DNA replication is probably carried out by a multiprotein complex containing numerous enzymes and accessory factors that act in concert to effect processive DNA synthesis (Applegren, N. et al. (1995) J. Cell. Biochem. 59, 91-107). We have exposed early zebrafish embryos to three chemical agents which are predicted to specifically inhibit the DNA polymerase alpha, topoisomerase I and topoisomerase II components of the DNA replication complex. We present four findings: (1) Before mid-blastula transition (MBT) an inhibition of DNA synthesis does not block cells from attempting to proceed through mitosis, implying the lack of functional checkpoints. (2) After MBT, the embryo displays two distinct modes of intrinsic checkpoint operation. One mode is a rapid and complete stop of cell division, and the other is an 'adaptive' response in which the cell cycle continues to operate, perhaps in a 'repair' mode, to generate daughter nuclei with few visible defects. (3) The embryo does not display a maximal capability for the 'adaptive' response until several hours after MBT, which is consistent with a slow transcriptional control mechanism for checkpoint activation. (4) The slow activation of checkpoints at MBT provides a window of time during which inhibitors of DNA synthesis will induce cytogenetic lesions without killing the embryo. This could be useful in the design of a deletion-mutagenesis strategy.

There have been many recent theoretical and technical advances in the sequencing of DNA in ultrathin slab gels. These include recent experimental progress in four areas: DNA polymerases; DNA template preparation; the separation and detection of DNA bands; and base-calling algorithms. The Zimm-Lumpkin theory of electrophoresis provides a new and improved framework for understanding the operation of sequencing gels.

We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 microns thick. Such a flattened whole mount allows imaging with a spatial resolution of approximately 500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10,000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.

Specific and processive antitermination by bacteriophage lambda N protein in vivo and in vitro requires the participation of a large number of Escherichia coli proteins (Nus factors), as well as an RNA hairpin (boxB) within the nut site of the nascent transcript. In this study we show that efficient, though nonprocessive, antitermination can be induced by large concentrations of N alone, even in the absence of a nut site. By adding back individual components of the system, we also show that N with nut+ nascent RNA is much more effective in antitermination than is N alone. This effect is abolished if N is competed away from the nut+ RNA by adding, in trans, an excess of boxB RNA. The addition of NusA makes antitermination by the N-nut+ complex yet more effective. This NusA-dependent increase in antitermination is lost when delta nut transcripts are used. These results suggest the formation of a specific boxB RNA-N-NusA complex within the transcription complex. By assuming an equilibrium model, we estimate a binding constant of 5 x 10(6) M-1 for the interaction of N alone with the transcription complex. This value can be used to estimate a characteristic dissociation time of N from the complex that is comparable to the dwell time of the complex at an average template position, thus explaining the nonprocessivity of the antitermination effect induced by N alone. On this basis, the effective dissociation rate of N should be approximately 1000-fold slower from the minimally processive (100-600 bp) N-NusA-nut+ transcription complex and approximately 10(5)-fold slower from the maximally processive (thousands of base pairs) complex containing all of the components of the in vivo N-dependent antitermination system.

Automated, direct cycle sequencing of purified double-stranded PCR products using Taq polymerase and fluorescently labeled dideoxynucleotide terminators provides a robust and highly reproducible method for identifying DNA sequence variations in sequence-tagged sites. We describe a simple and sensitive strategy that reliably detects the presence of DNA variations when sequencing traces from several different individuals are compared. We also demonstrate the use of this strategy to estimate allele frequencies of single nucleotide substitutions in a population. Taken together, this approach provides an automated method for conducting rapid population studies of candidate gene regions that are in linkage or association with a specific disease and for comparative evolutionary analysis of selected regions of the human genome.

The capacity of the globular domain of the chicken erythrocyte linker histone H5 (GH5) to self-associate in solution has been demonstrated by chemical cross-linking with dimethyl 3,3'-dithiobis-(propionimidate) (DTBP), dithiobis(succinimidyl propionate) (DSP), and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP). Several observations suggest that the GH5-GH5 interactions that mediate self-association are specific: (a) Incubation with each of the above reagents produces a discrete and characteristic pattern of cross-linked products; (b) GH1, the related peptide from chicken erythrocyte H1, is not cross-linked under the same conditions; (c) GH5 is not cross-linked with disuccinimidyl tartrate (DST), which has a shorter cross-linking span (6.4 A) than the other reagents (12 A); and (d) analysis of cross-linking as a function of peptide concentration provides an equilibrium constant for GH5 self-association of (4.8 +/- 1.3) x 10(3) M-1. The ability of GH5 to specifically self-associate is compatible with the proposal [Thoma, F., Koller, T., & Klug, A. (1979) J. Cell Biol. 83, 403-427] that linker histone globular domains occupy an axial position within the higher order chromatin fiber; the spatial juxtaposition of the GH5 domains at this location would be expected to promote their association and exert a stabilizing effect upon higher order chromatin structure.

We show that the amino acid analogue betaine shares with small tetraalkylammonium ions [Melchior, W. B., Jr., & von Hippel, P. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 298-302] the ability to reduce or even eliminate the base pair composition dependence of DNA thermal melting transitions. The "isostabilizing" concentration of betaine (at which AT and GC base pairs are equally stable) is approximately 5.2 M. Betaine exerts its isostabilizing effect without appreciably altering the conformation of double-stranded DNA from the B form. The presence of > 5 M betaine also does not greatly change the behavior of DNA as a polyelectrolyte; this lack of effect on electrostatic interactions is expected because betaine exists as a zwitterion near neutral pH. Study of DNA melting transitions in high concentrations of betaine thus allows the experimental separation of compositional and polyelectrolyte effects on DNA melting. As a consequence, betaine solutions can also be used to investigate DNA-protein interactions under isostabilizing (or close to isostabilizing) conditions, which has not been possible using isostabilizing salts. This potential is illustrated by examining the highly salt concentration-dependent interaction of ribonuclease A with DNA in concentrated betaine solutions.

The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n > or = 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds approximately 70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.

At any template position, the decision to extend the transcript by one residue or to release the nascent RNA represents a kinetic competition between elongation and termination pathways. This competition is discussed in terms of alternative Eyring transition state barriers; changes in termination efficiency correspond to small changes in the relative heights of these barriers. Elongation complexes are stable at nonterminator positions; a model is presented to explain the destabilization of these complexes at intrinsic termination sites. Functionally analogous effects can operate at rho-dependent terminators. Mechanisms for modulation of termination efficiency by regulatory proteins are described.

To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex. To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers. Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment. The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance. Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A. The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A. These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy. In the following paper in this issue [Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C., & von Hippel, P. H. (1992)], these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.

Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry. Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.

This review has summarized the known features of the single-nucleotide addition reaction cycle in transcription. The reader will have noted that the information available is very incomplete, and that, in some cases, related experiments seem to lead to contradictory conclusions. We have tried to point out these discrepancies as they occur and to indicate areas where more experimentation is needed. We look forward to the day when all the microscopic steps of the single-nucleotide addition cycle can be identified and defined in thermodynamic, kinetic, and structural terms. At that point, we can begin to understand the principles that relate these parameters to template position and to the pathway of formation of a specific complex. It should be possible to provide specific molecular interpretations for observed effects on activation barrier heights to elongation and termination (154, 155) and to begin to understand the molecular bases of the regulation in these phases of transcription. Much work remains before this happy situation can be totally realized, but we feel that now the problem can at least be approached at this level. We hope that this review helps to illuminate the difficulties that remain.

The Human Genome Project (HGP) is an international effort to map and sequence the human genome. It combines skills from diverse fields of biological and technological research, thus establishing deeper interactions between scientific disciplines. The combination of these skills should stimulate many advances in both pure and applied fields of research and give rise to new, interdisciplinary training programs. Some critics say that the HGP will damage biomedical research; however, we argue that it will bring new funds to the field and create a large ripple effect by providing new research opportunities through its discoveries.

In this paper we examine the physical properties and potential for self-association of the Escherichia coli transcription factors, sigma 70 and NusA. We show, by a combination of chemical crosslinking, equilibrium and velocity sedimentation, quasi-elastic light scattering, and small-angle X-ray scattering that NusA exists as a monomer at KCl concentrations between 0.01 and 1.5 M, and that sigma 70 exists as a monomer at KCl concentrations between 0.1 and 1.5 M. The shape and hydration characteristics of each of these monomeric proteins are also examined. The results serve as background for the companion paper in which a thermodynamic analysis is made of the interactions of these transcription factor with E. coli core RNA polymerase in solution and as a component of the functional transcription complex.

In this paper, we develop a kinetic approach to predict the efficiency of termination at intrinsic (factor independent) terminators of Escherichia coli and related organisms. In general, our predictions agree well with experimental results. Our analysis also suggests that termination efficiency can readily be modulated by protein factors and environmental variables that shift the kinetic competition toward either elongation or termination. A quantitative framework for the consideration of such regulatory effects is developed and the strengths and limitations of the approach are discussed.

In the first part of this paper we present a thermodynamic analysis of the elongation phase of transcription in Escherichia coli. The stability of the elongation complex is described by a "free energy of formation" function (delta G zero f) that is a sum of terms for forming (i) a locally denatured 17-base-pair DNA "bubble"; (ii) a constant-length hybrid between the 3'-terminal 12-nucleotide residues of the RNA transcript and the corresponding region of the DNA template strand; and (iii) a set of binding interactions between the polymerase and certain DNA and RNA residues within and near the "transcription bubble". The transcriptional elongation complex is very stable at most positions along a natural DNA template and moves in a highly processive fashion. At these positions, the delta G zero f function provides a quantitative measure of the stability of the elongation complex. Besides allowing for the polymerization of the RNA transcript, the elongation complex also serves to define the context within which transcript termination occurs. In the second part of the paper the thermodynamic analysis is extended to discriminate between template positions at which the elongation complex is stable and positions at which it is rendered relatively unstable by the presence of a string of rU residues at the 3'-terminus of the RNA together with the formation of a specific RNA hairpin just upstream of this point. Most factor-independent (intrinsic) termination events are thermodynamically disallowed at the former positions and are thermodynamically allowed at the latter positions. The extended form of the analysis closely predicts the exact sites of termination at a number of intrinsic terminators (and attenuators) in the E. coli genome. It also correctly predicts bidirectional function for a number of bidirectional terminators. In some cases it may identify terminators that are similar to the intrinsic type but that require additional protein factors, unusual polymerase-nucleic acid interactions, or rate-limiting conformational changes in order to function. Finally, it successfully locates intrinsic terminators within a number of E. coli operons and discriminates between these terminators and the surrounding DNA sequence.

The E. coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination. In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex. The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process. This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete.

By two independent methods, we have determined approximately the time-course of hatching enzyme secretion in the sea urchin Strongylocentrotus purpuratus. A quick-kill method indicates that a significant fraction of the enzyme is secreted between 90% and 97% of the fertilization-hatching interval. A direct assay method indicates that the remainder of the enzyme is secreted on either side of the 90-97%"window". The entire period of secretion spans from 75% to 100% or more of the fertilization-hatching interval. For embryos raised at 15°C this translates to an interval of 4.8 or more hr.

We have prepared chicken erythrocyte nucleosomes lacking proteins other than the inner histones and containing long DNA. Our nucleosomes' DNA has mean length +/- SD = 190 +/- 15 base pairs. No DNA less than 155 base pairs is present. Nucleosome stability in salt was examined by boundary and band sedimentation and by particle gel electrophoresis. We find the following. (i) A second species is slowly generated by treatment with salt. This species sediments with S20,w = 5.5 S (as does purified mononucleosomal DNA), is not associated with histones, and electrophoretically migrates as mononucleosomal DNA. We conclude it is free DNA. Thus, salt causes nucleosomes to dissociate, independently of either noncore proteins, or of any nucleosome population with DNA less than 146 base pairs. (ii) Dissociation is reversible, and is enhanced by nucleosome dilution. Thus, it appears to follow the law of mass action. (iii) The equilibrium extent of dissociation increases with salt. A second effect of salt is a fast, reversible 10% decrease in S20,w of the nucleosomes left intact. From hydrodynamic calculations, this is consistent with either a slight unfolding of the entire nucleosome, or an unbinding of the terminal DNA regions from the histone core.

At elevated salt concentrations, the structure of chromatin is destabilized. This paper is concerned with the processes by which DNA is released from nucleosome core particles in free, uncomplexed form. Our experiments indicate that the DNA release reaction has distinctly different characteristics below and above approximately 0.75 M NaCl. Below this concentration of salt, release of the histones from the DNA is highly cooperative, so that no dissociation intermediates are even seen. Above this salt concentration, histone release is not so cooperative; H2A and H2B are released from the DNA more readily than are H3 and H4. This results in an apparently heterogeneous population of (H2A, H2B)-depleted intermediate species sedimenting at rates between that of free DNA and that of intact core particles. Dissociation of core particles at NaCl concentrations below 0.75 M is readily reversible. Reassociation of DNA and histones from higher salt concentrations is nearly quantitative if carried out by gradual decrease of salt concentration, but rapid dilution to low salt results in the formation of a fraction of metastable nucleosome multimers. To help organize our description of the DNA release process, we introduce a stability diagram for the core particle, defined with respect to the independent variables of salt concentration and particle concentration. We draw upon our own experimental work and also upon the work of several other laboratories. We distinguish five major regions in this diagram.