Post-embryonic expression of C. elegans microRNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system.

MicroRNAs (miRNAs) are regulatory molecules that negatively control gene expression by binding to complementary sequences on target mRNAs. The most thoroughly characterized miRNAs, lin-4 and let-7, direct cell fate determination during the larval transitions in C. elegans and act as key regulators of temporal gene expression. lin-4 and let-7 are founding members of two distinct families of miRNA genes sharing strong sequence homology primarily in the 5' end of the mature miRNAs. In this report, we characterize the temporal and spatial expression patterns of lin-4 and let-7 family members using northern blot analysis and mir::gfp fusion studies. Our results show that lin-4 and let-7 homologues possess distinct temporal and spatial expression patterns during nematode development and that known heterochronic genes regulate their expression. We find that certain lin-4 and let-7 family members display overlapping expression patterns in the hypodermis and the reproductive system, suggesting that combinations of miRNAs from across families may control common developmental events.

Imaging parallel fiber and climbing fiber responses and their short-term interactions in the mouse cerebellar cortex in vivo.

A major question in the study of cerebellar cortical function is how parallel fiber and climbing fiber inputs interact to shape information processing. Emphasis has been placed on the long-term effects due to conjunctive stimulation of climbing fibers and parallel fibers. Much less emphasis has been placed on short-term interactions and their spatial nature. To address this question the responses to parallel fiber and climbing fiber inputs and their short-term interaction were characterized using optical imaging with Neutral Red in the anesthetized mouse in vivo. Electrical stimulation of the cerebellar surface evoked an increase in fluorescence consisting of a transverse optical beam. The linear relationship between the optical responses and stimulus parameters, high spatial resolution and close coupling to the electrophysiological recordings show the utility of this imaging methodology. The majority of the optical response was due to activation of postsynaptic alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) and metabotropic glutamate receptors with a minor contribution from the presynaptic parallel fibers. Stimulation of the inferior olive evoked parasagittal bands that were abolished by blocking AMPA glutamate receptors. Conjunctive stimulation of the cerebellar surface and inferior olive resulted in inhibition of the climbing fiber evoked optical responses. This lateral inhibition of the parasagittal bands extended out from both sides of an activated parallel fiber beam and was mediated by GABA(A) but not GABA(B) receptors. One hypothesized role for lateral inhibition of this type is to spatially focus the interactions between parallel fiber and climbing fiber input on Purkinje cells. In summary optical imaging with Neutral Red permitted visualization of cerebellar cortical responses to parallel fiber and climbing fiber activation. The GABA(A) dependent lateral inhibition of the climbing fiber evoked parasagittal bands by parallel fiber stimulation shows that cerebellar interneurons play a short-term role in shaping the responses of Purkinje cells to climbing fiber input.

Design of a compartmentalized shotgun assembler for the human genome.

Two different strategies for determining the human genome are currently being pursued: one is the "clone-by-clone" approach, employed by the publicly funded project, and the other is the "whole genome shotgun assembler" approach, favored by researchers at Celera Genomics. An interim strategy employed at Celera, called compartmentalized shotgun assembly, makes use of preliminary data produced by both approaches. In this paper we describe the design, implementation and operation of the "compartmentalized shotgun assembler".

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

An iterative method for faster sum-of-pairs multiple sequence alignment.

MOTIVATION: Multiple sequence alignment is an important tool in computational biology. In order to solve the task of computing multiple alignments in affordable time, the most commonly used multiple alignment methods have to use heuristics. Nevertheless, the computation of optimal multiple alignments is important in its own right, and it provides a means of evaluating heuristic approaches or serves as a subprocedure of heuristic alignment methods. RESULTS: We present an algorithm that uses the divide-and-conquer alignment approach together with recent results on search space reduction to speed up the computation of multiple sequence alignments. The method is adaptive in that depending on the time one wants to spend on the alignment, a better, up to optimal alignment can be obtained. To speed up the computation in the optimal alignment step, we apply the alpha(*) algorithm which leads to a procedure provably more efficient than previous exact algorithms. We also describe our implementation of the algorithm and present results showing the effectiveness and limitations of the procedure.

A whole-genome assembly of Drosophila.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

The practical use of the A* algorithm for exact multiple sequence alignment.

Multiple alignment is an important problem in computational biology. It is well known that it can be solved exactly by a dynamic programming algorithm which in turn can be interpreted as a shortest path computation in a directed acyclic graph. The A* algorithm (or goal-directed unidirectional search) is a technique that speeds up the computation of a shortest path by transforming the edge lengths without losing the optimality of the shortest path. We implemented the A* algorithm in a computer program similar to MSA (Gupta et al., 1995) and FMA (Shibuya and Imai, 1997). We incorporated in this program new bounding strategies for both lower and upper bounds and show that the A* algorithm, together with our improvements, can speed up computations considerably. Additionally, we show that the A* algorithm together with a standard bounding technique is superior to the well-known Carrillo-Lipman bounding since it excludes more nodes from consideration.

DNA multimode interaction with berenil and pentamidine; double helix stiffening, unbending and bending.

The antitrypanosomal drugs berenil (Ber) and pentamidine (Pm) preferentially bind to DNA in the minor groove of A.T-rich domains. The properties of A.T clusters are essential for sequence-mediated helix bending. Groove binding drugs locally stiffen the DNA helix but may also change intrinsic helix bends or may bend straight DNA. Ligand binding to randomly distributed sites alters the apparent DNA persistence length, a. Criteria permit the distinction of the underlying mechanism(s). Helix bends, if phased with the helix screw, however, generate solenoidal superhelix components mediating an apparent change of the hydrodynamically effective DNA contour length, L. The measurement of relative changes of both, a and L, as induced by Ber or Pm is performed by titration rotational viscometry. The determination of the two quantities requires two independent measurements: the relative change of DNA intrinsic viscosity, deltay, for short (tending to rod-like) DNA molecules and for comparably long (almost coil-like) ones as a function of r, the bound drug molecules per DNA-P, and this under conditions effectively excluding intramolecular DNA-DNA crosslinking effects. At least at r< or =0.05 and < or =0.03, respectively, the two drugs virtually bind completely to a eukaryotic DNA. r ranges of different drug binding strength and, concomitantly, of different specific conformational response, could be resolved. They represent (sub)modes of different DNA sequences... Whereas the mode-specific elongation effects are fairly similar for both systems, there are pronounced quantitative differences in the relative change of DNA persistence length. The sites of highest Ber-binding strength are correlated to unbent alternating helical A.T segments followed by bent and by less bent or unbent dAn.dTn tracts straightened on Ber-binding. For Pm-DNA interaction the ligand bends the sites of highest Pm affinity. Generally, ligand induced and sequence mediated local DNA-bend removal or DNA bending, as observed for several modes of interaction with A.T rich DNA, are considered to be of gene regulatory relevance.

An exact solution for the segment-to-segment multiple sequence alignment problem.

MOTIVATION: In molecular biology, sequence alignment is a crucial tool in studying the structure and function of molecules, as well as the evolution of species. In the segment-to-segment variation of the multiple alignment problem, the input can be seen as a set of non-gapped segment pairs (diagonals). Given a weight function that assigns a weight score to every possible diagonal, the goal is to choose a consistent set of diagonals of maximum weight. We show that the segment-to-segment multiple alignment problem is equivalent to a novel formulation of the Maximum Trace problem: the Generalized Maximum Trace (GMT) problem. Solving this problem to optimality, therefore, may improve upon the previous greedy strategies that are used for solving the segment-to-segment multiple sequence alignment problem. We show that the GMT can be stated in terms of an integer linear program and then solve the integer linear program using methods from polyhedral combinatorics. This leads to a branch-and-cut algorithm for segment-to-segment multiple sequence alignment. RESULTS: We report on our first computational experiences with this novel method and show that the program is able to find optimal solutions for real-world test examples.

A polyhedral approach to RNA sequence structure alignment.

Ribonucleic acid (RNA) is a polymer composed of four bases denoted A, C, G, and U. It generally is a single-stranded molecule where the bases form hydrogen bonds within the same molecule leading to structure formation. In comparing different homologous RNA molecules it is important to consider both the base sequence and the structure of the molecules. Traditional alignment algorithms can only account for the sequence of bases, but not for the base pairings. Considering the structure leads to significant computational problems because of the dependencies introduced by the base pairings. In this paper we address the problem of optimally aligning a given RNA sequence of unknown structure to one of known sequence and structure. We phrase the problem as an integer linear program and then solve it using methods from polyhedral combinatorics. In our computational experiments we could solve large problem instances--23S ribosomal RNA with more than 1400 bases--a size intractable for former algorithms.

Multimode interaction of Hoechst 33258 with eukaryotic DNA; quantitative analysis of the DNA conformational changes.

The interaction of the minor groove binding ligand Hoechst 33258 (Hoe) with natural DNA was investigated by high resolution titration rotational viscometry. Analysis of the concomitant DNA conformational changes was performed with two DNA samples of sufficiently different molar mass M, at 4 degrees C, 22 degrees C and 40 degrees C, for Hoe/DNA-P ratios below r = 0.02. In this narrow r range several interaction modes could be resolved. The measured conformational changes were quantified in terms of relative changes of both apparent DNA persistence length, delta a/a, and hydrodynamically operative DNA contour length, deltaL/L. Delta a/a(r) primarily is a measure of ligand-induced DNA helix stiffening, but both, delta a/a(r) and deltaL/L(r), generally depend also on ligand binding induced DNA bending or DNA unbending. The essential difference obviously is that delta a/a(r) is influenced by the randomly distributed helix bends and deltaL/L(r) by phased ones. The measurements performed at different temperatures deliver informations about existence and temperature dependent abolition of intrinsic helix curvature. Both Hoe and netropsin (Nt) prefer binding to AT rich DNA segments, which are candidates for intrinsic DNA helix bends. But our data for Hoe interaction with calf thymus DNA (ctDNA) show characteristic differences to those for Nt-ctDNA interaction. Especially for Hoe, the mode of highest affinity is saturated already at a ligand concentration of roughly 1 nM (r approximately = 0.0015 Hoe/DNA-P). It exhibits an unusually strong temperature dependence of the conformational DNA response. A Hoe-Nt competition experiment shows that Hoe binding to the sites of the very first Hoe mode is almost unaffected by bound Nt. But Hoe binding to the sites of the following Hoe modes does not occur due to the competition with Nt. Thus this mode of strongest Hoe-DNA interaction reflects a unique mechanism, possibly of high relevance for gene regulatory systems.

Deformyldistamycin-DNA interaction; DNA conformational changes as revealed by titration rotational viscometry.

The conformational changes of a natural DNA species on binding of deformyl-distamycin (dDst) have been analysed, at 22 degrees C and 7.6 degrees C, in terms of changes of apparent persistence length (a) and of apparent contour length (L), by means of titration rotational viscometry with both high and low molar mass calf thymus (ct) DNA molecules. Next to ligand binding mediated alterations in DNA stiffness, changes of a are the result of helix bending and also of unbending of intrinsic helix bends. A test for the latter are viscosity measurements at different temperatures. Changes of L, on binding of non-intercalating ligands, are interpreted as the result of changes in the intrinsic solenoidal structure components of the natural eukaryotic DNA. Such tertiary-structure components do exist if base sequence dependent helix bends of (nucleosomal) DNA are phased with the helix screw [Drew & Travers, JMB 186, 773 (1985); Reinert et al., JBSD 9, 537 (1991)]. Hence, the measured very small changes of L at ligand/DNA-P ratios r < 0.02 are mainly understood as a partial abolition of intrinsic tertiary structure components and the following negative ones as a respective reinforcement of such structures by dDst binding to AT rich binding sites. Several r-intervals with different slope of the viscosity changes could be resolved at r < 0.05. The resolution of more than four modes of dDst interaction with ctDNA at very low r values is comparable to DNA interaction of Nt and several other ligands but not of distamycin. Advanced titration rotational DNA-viscometry is again able to resolve subtle quantitative details of ligand mediated DNA conformational changes of high stereochemical relevance.

Aquatic risk assessment of a polycarboxylate dispersant polymer used in laundry detergents.

Polycarboxylates enhance detergent soil removal properties and prevent encrustation of calcium salts on fabrics during washing. Laundry wastewater typically reaches wastewater treatment plants, which then discharge into aquatic environments. The yearly average concentration of a 4500 molecular weight (MW) sodium acrylate homopolymer reaching U.S. wastewater treatment plants will be approximately 0.7 mg/L. Publications showing the low to moderate acute aquatic toxicity of polycarboxylates are readily available. However, there are no published evaluations that estimate wastewater removal and characterize the probability of exceedance of acceptable chronic aquatic exposure. WW-TREAT can be used to estimate removal during wastewater treatment and PG-GRIDS can be applied to characterize risk for exceedance in wastewater treatment plant outfalls. After adjustments for the MW distribution of the homopolymer, WW-TREAT predicted that 6.5% will be removed in primary treatment plants and 60% will be removed in combined primary and activated sludge treatment plants. These estimates are consistent with wastewater fate tests, but underestimate homopolymer removal when homopolymer precipitation is included. Acceptable levels of chronic outfall (receiving water) exposure were based on aquatic toxicity testing in algae, fish, and Daphnia magna. PG-GRIDS predicted that no unreasonable risk for exceedance of acceptable chronic exposure will occur in the outfalls of U.S. wastewater plants. Future development of wastewater treatment models should consider polymer MW distribution and precipitation as factors that may alter removal of materials from wastewater.

DNA interaction of the imidazole-containing lexitropsin ImPy: titration viscometric study in comparison to netropsin.

The imidazole (Im) containing lexitropsin ImPy related to netropsin (Nt) is a sequence reading DNA ligand which, in contrast to Nt, permits binding to a GC base pair. The ImPy induced DNA conformational changes differ significantly from those induced by Nt as monitored by titration viscometry, although interaction modes have also been resolved with boundaries at the same ligand to DNA phosphate ratio, r. Evidently ImPy covers similar binding sites (in the same sequence) as Nt for natural calf thymus DNA at r < 0.023. This result suggests that the preferred binding sites of ImPy are A tracts (cf. K.E.R. JBSD 9(1993) 973), in agreement with previous data. The respective DNA coil expansion, most probably caused by unbending (l.c.), is similar but smaller compared to the Nt-DNA interaction. These results again suggest that, at low r values, the van der Waals interaction in the narrowed minor groove of AT clusters provides a dominating energy contribution to ImPy binding. At r > 0.03 the DNA coil expansion increases to extremely high values in that r range where Nt binding (to mixed AT/GC sequences) induces no effect at all owing to steric hindrance with the amino group of guanine. On the basis of many quantitative results for the Nt-DNA systems these effects can be understood in terms of an unbending of intrinsic helix bends (l.c.). They are of considerable interest in connection with the ability of such compounds to influence the direction of the local regulatory relevant DNA curvature.

Polymers as Solid Waste in Municipal Landfills.

Synthetic polymers reach municipal landfills as components of products such as waste household paints, packaging films, storage containers, carpet fibers, and absorbent sanitary products. Some polymers in consumer products that reach landfills are designed to photodegrade or biodegrade. This article examines the significance of degradable polymers in management of solid waste in municipal landfills. Most landfills are not designed to photodegrade or biodegrade solid waste. Landfill disposal of stable polymers such as polyacrylics and polyethylenes is not associated with significant polymer degradation or mobility. Stability to photodegradation and biodegradation is an advantage when municipal landfills are used for disposal of polymer products as solid waste. Use of landfill disposal can be a responsible means to manage polymer waste and can be part of an overall waste management plan which includes source reduction, recycling, reuse, composting, and waste-to-energy incineration.

T helper cell alveolitis after bacillus Calmette-Guerin immunotherapy for superficial bladder tumor.

We report a case of T helper cell alveolitis after intravesical bacillus Calmette-Guerin (BCG) immunotherapy for a superficial bladder tumor. Despite the clinical appearance of miliary pulmonary tuberculosis no mycobacterial agent was found. Therefore, we concluded that the patient had a hypersensitivity reaction similar to sarcoidosis. The condition improved after immunosuppressive therapy with glucocorticosteroids. Differentiation of bronchoalveolar cells could be useful in distinguishing hypersensitivity to infection with BCG.

Abolition of intrinsically bent DNA structure components in AT clusters by netropsin interaction; titration viscometric investigations.

It is argued that the enhancement of the apparent DNA contour length by the specifically binding non-intercalating drug netropsin (Nt) (Reinert et al., NAR 9, 2335, 1981) at very low Nt/DNA-phosphate ratios essentially is the result of an abolition of periodically arranged intrinsic helix bends in A.T rich tracts of base pairs. In the preceding paper the existence of pronounced DNA tertiary structure components has been postulated for (two species of) natural eukaryotic DNA. The resulting model suggests local apparent solenoid-related DNA tertiary structure components at high sodium ion concentration cs, partly/totally molten out at 45/60 C. With decreasing cs the tertiary structure components have been found to be gradually reduced, at least below cs = 0.010 M, as titration viscometrically revealed by a gradual rise of the apparent DNA contour length (Reinert et al., JBSD 9, 537, 1991). Hence, we performed titration viscometric analyses about Nt interaction with calf thymus DNA (ctDNA) at cs = 0.075 M, 0.010 M and 0.004 M Na+. The concomitant DNA conformational changes are quantitatively described in terms of the relative changes of both DNA persistence length and hydrodynamically operative apparent DNA contour length for the three first resolved interaction modes below a Nt/DNA-P ratio of 0.03. These experiments, together with previous respective analyses at cs = 0.20 M Na+ and different temperatures (l.c.), suggest that those DNA sites binding Nt most strongly predominantly are responsible for the formation of solenoid-related DNA tertiary structure components. Most probably these are A tract-containing sequences. As the essential factor for their apparent elongation effect at low Na+ concentrations, a gradual alteration of the number of base pairs per helix turn seems to occur below cs = 0.010 M Na+ and, concomitantly, a change in phasing between intrinsic helix bends and helix screw.

Counterion-type characteristic effects on intrinsic bending components of calf thymus DNA; hydrodynamic investigations.

This paper stresses structural differences in A.T clusters of the ammonium salt of calf thymus (ct) DNA (ctNH4DNA) and the respective sodium salt, ctNaDNA. Sequence mediated intrinsic helix bends of ctNaDNA, distributed along the molecule partially randomly and partially phased with the helix screw (accompanying paper), are enhanced in ctNH4DNA. Additionally, the number of the most strongly bent segments (of A-tract character) is raised in ctNH4DNA by a counterion mediated shift of the equilibrium between at least two local DNA conformations. Nevertheless, the apparent DNA elongation, induced by the abolition of a single apparent solenoid-related DNA tertiary structure component which generates a special intrinsic DNA bend, is the same for NH4DNA and NaDNA. These conclusions follow from two independent sets of experimental results: (1.) Titration viscometric measurements with ctNH4DNA as a function of the cation concentration in comparison to ctNaDNA (KER et al., JBSD 9,537 (1991)) and respective DNA conformational analyses. (2.) Quantitative viscometric analysis of DNA conformational changes on netropsin (Nt) interaction of ctNH4DNA at different temperatures and comparison with the respective data for ctNaDNA (KER et al., NAR 9,2335 (1981).

Accuracy of self-reported health histories: a study.

Many military health care providers (including most dental providers) depend on self-reported health questionnaires for critical information about their patients' medical history. These questionnaires demand high standards of patient self-awareness and integrity, and their importance justifies checking their accuracy. The authors checked the accuracy of 155 self-reported health histories by comparing them with histories documented in medical records. Although we found some discrepancies, over 95% of our sample showed reasonable agreement between self-reported medical histories and documented medical histories.