The elongation-termination decision in transcription.

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.

The Human Genome Project: creating an infrastructure for biology and medicine.

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.

A Four-Biomarker Blood Signature Discriminates Systemic Inflammation Due to Viral Infection Versus Other Etiologies.

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.

Escherichia coli sigma 70 and NusA proteins. II. Physical properties and self-association states.

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.

High-speed DNA sequencing in ultrathin slab gels.

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.

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA.

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.

Thermodynamic analysis of the transcription cycle in E. coli.

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.

Blood-based biomarkers for detecting mild osteoarthritis in the human knee.

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.

Preparation and imaging of nuclear spreads from cells of the zebrafish embryo. Evidence for large degradation intermediates in apoptosis.

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.

Developmental activation of the capability to undergo checkpoint-induced apoptosis in the early zebrafish embryo.

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.

Dynamics and equilibria of nucleosomes at elevated ionic strength.

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.

Transcript elongation and termination are competitive kinetic processes.

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.

A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli.

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.

Self-association of the globular domain of histone H5.

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.

Improvement of base-calling in multilane automated DNA sequencing by use of electrophoretic calibration standards, data linearization, and trace alignment.

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.

Comparative analysis of human DNA variations by fluorescence-based sequencing of PCR products.

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.

Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer.

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.

Physical properties of the Escherichia coli transcription termination factor rho. 2. Quaternary structure of the 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.

Betaine can eliminate the base pair composition dependence of DNA melting.

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.