[The epidemiology and clinical features of Legionella pneumonia (LP) in patients older than 60 years old who were hospitalized with pneumonia in northern Israel].

BACKGROUND: Risk factors associated with LP are frequent in patients older than 60 years old who are hospitalized with pneumonia. The aim of the study was to define the incidence, epidemiological and clinical features of LP in this age group in Northern Israel. STUDY DESIGN: The study was prospective and conducted for one year during the period 1.6.1999-31.5.2000. All patients older than 60 years who were hospitalized with community-acquired or nosocomial pneumonia were tested for legionella infection by the urine antigen test (which identifies Legionella pneumophila type I and 14 other Legionella serotype antigens). Data was obtained from each patient regarding risk factors and clinical feature of the disease. The data of patients with LP was compared on a 1:2 ratio to data obtained from a control group of patients with non LP according to age, sex, and week of admission. RESULTS: During the study period 202 patients and 38 patients were hospitalized with community-acquired or nosocomial pneumonia respectively. Overall, 8/240 patients (3.3%) were found to suffer from LP. All patients with LP had community-acquired pneumonia with an incidence of 8/202 (4%). Six of the 8 patients (75%) with LP were hospitalized during June-September. Significant clinical findings in patients with LP as compared to those in the control group, respectively, were: severity score, history of smoking, mental status alteration, respiration rate over 30/minute, respiratory acidosis, hypoxia, and need for mechanical ventilation (P < 0.05 in all). All patients with LP were treated with macrolides, however the death rate was 50% vs 0% in the control group (p < 0.001). CONCLUSIONS: In northern Israel, LP is infrequent among patients older than 60 years hospitalized with pneumonia. The disease occurs mostly during the summer in patients with community acquired pneumonia. Patients with LP had unique and more severe clinical features and the death rate was very high inspite of appropriate therapy.

The hepatitis B virus pregenome: prediction of RNA structure and implications for the emergence of deletions.

The terminally redundant pregenomic RNA of human hepatitis B virus (HBV) comprises some 3,330 nucleotides and is a replicative intermediate in the production of the circular DNA genome. Deletions are known to arise in the HBV genome during the course of chronic infection and are sometimes associated with interferon therapy. These deletions are limited to small parts of the genome such as the 357-nucleotide pre-S1 region. Long RNA molecules such as the HBV pregenome have considerable structural flexibility and will undergo secondary structure shifts between energetically favourable states in a continuous and semi-random fashion. Since prediction of structure elements that are highly conserved in different forms of one RNA molecule is now feasible by computer modelling, we have analysed the whole HBV pregenome by two different RNA structure prediction algorithms and by new methods that exploit these algorithms. Significantly, the ends of pregenomic RNA were predicted to undergo both short-range and long-range interactions, which has relevance to our knowledge of the virus replicative cycle. By incorporating phylogenetic information relating to the 6 recognised genotypes of HBV, it was possible to highlight short secondary structures that may be common to all HBV strains. For example, although the pre-S1 region was predicted to undergo local folding of a loosely defined nature, most observed pre-S1 deletions mapped to all or part of an arm carrying a better-defined structure. The loss of such sequences may be mechanistically attributable to polymerase skipping during reverse transcription, and the possible advantages of such deletions are considered.

Metrics on RNA secondary structures.

Many different programs have been developed for the prediction of the secondary structure of an RNA sequence. Some of these programs generate an ensemble of structures, all of which have free energy close to that of the optimal structure, making it important to be able to quantify how similar these different structures are. To deal with this problem, we define a new class of metrics, the mountain metrics, on the set of RNA secondary structures of a fixed length. We compare properties of these metrics with other well known metrics on RNA secondary structures. We also study some global and local properties of these metrics.

Calculating nucleic acid secondary structure.

New results for calculating nucleic acid secondary structure by free energy minimization and phylogenetic comparisons have recently been reported. A complete set of DNA energy parameters is now available and the RNA parameters have been improved. Although databases of RNA secondary structures are still derived and expanded using computer-assisted, ad hoc comparative analysis, a number of new computer algorithms combine covariation analysis with energy methods.

Fast evaluation of internal loops in RNA secondary structure prediction.

MOTIVATION: Though not as abundant in known biological processes as proteins, RNA molecules serve as more than mere intermediaries between DNA and proteins. Research in the last 15 years demonstrates that RNA molecules serve in many roles, including catalysis. Furthermore, RNA secondary structure prediction based on free energy rules for stacking and loop formation remains one of the few major breakthroughs in the field of structure prediction, as minimum free energy structures and related quantities can be computed with full mathematical rigor. However, with the current energy parameters, the algorithms used hitherto suffer the disadvantage of either employing heuristics that risk (though highly unlikely) missing the optimal structure or becoming prohibitively time consuming for moderate to large sequences. RESULTS: We present a new method to evaluate internal loops utilizing currently used energy rules. This method reduces the time complexity of this part of the structure prediction from O(n4) to O(n3), thus reducing the overall complexity to O(n3). Even when the size of evaluated internal loops is bounded by k (a commonly used heuristic), the method presented has a competitive edge by reducing the time complexity of internal loop evaluation from O(k2n2) to O(kn2). The method also applies to the calculation of the equilibrium partition function. AVAILABILITY: Source code for an RNA secondary structure prediction program implementing this method is available at ftp://www.ibc.wustl.edu/pub/zuker/zuker .tar.Z

Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure.

An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodynamic parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent experiments. Additional algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures? determined by comparative sequence analysis was assembled to allow optimization of parameters not based on experiments and to test the accuracy of the algorithm. On average, the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on average, has 86 % of known base-pairs. Experimental constraints, derived from enzymatic and flavin mononucleotide cleavage, improve the accuracy of structure predictions.

Using reliability information to annotate RNA secondary structures.

A number of heuristic descriptors have been developed previously in conjunction with the mfold package that describe the propensity of individual bases to participate in base pairs and whether or not a predicted helix is "well-determined." They were developed for the "energy dot plot" output of mfold. Two descriptors, P-num and H-num, are used to measure the level of promiscuity in the association of any given nucleotide or helix with alternative complementary pairs. The third descriptor, S-num, measures the propensity of bases to be single-stranded. In the current work, we describe a series of programs that were developed in order to annotate individual structures with "well-definedness" information. We use color annotation to present the information. The programs can annotate PostScript files that are created by the mfold package or the PostScript secondary structure plots produced by the Weiser and Noller program XRNA (Weiser B, Noller HF, 1995, XRNA: Auto-interactive program for modeling RNA, The Center for Molecular Biology of RNA, Santa Cruz, California: University of California; Internet: ftp://fangio.ucsc.edu/pub/XRNA). In addition, these programs can annotate ss files that serve as input to XRNA. The annotation package can also handle structure comparison with a reference structure. This feature can be used to compare predicted structure with a phylogenetically deduced model, to compare two different predicted foldings, and to identify conformational changes that are predicted between wild-type and mutant RNAs. We provide several examples of application. Predicted structures of two RNase P RNAs were colored with P-num information and further annotated with comparative information. The comparative model of a 16S rRNA was annotated with P-num information from mfold and with base pair probabilities obtained from the Vienna RNA folding package. Further annotation adds comparisons with the optimal foldings obtained from mfold and the Vienna package, respectively. The results of all of these analyses are discussed in the context of the reliability of structure prediction.

Structural plasticity in RNA and its role in the regulation of protein translation in coliphage Q beta.

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.

The Giardia lamblia actin gene and the phylogeny of eukaryotes.

The single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kineto-platid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to small-subunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching.

"Well-determined" regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA.

Recent structural analyses of genomic RNAs from RNA coliphages suggest that both well-determined base paired helices and well-determined structural domains that are identified by "energy dot plot" analysis using the RNA folding package mfold, are likely to be predicted correctly. To test these observations with another group of large RNAs, we have analyzed 15 ribosomal RNAs. Published secondary structure models that were derived by comparative sequence analysis were used to evaluate the predicted structures. Both the optimal predicted fold and the predicted "energy dot plot" of each sequence were examined. Each prediction was obtained from a single computer run on an entire ribosomal RNA sequence. All predicted base pairs in optimal foldings were examined for agreement with proven base pairs in the comparative models. Our analyses show that the overall correspondence between the predicted and comparative models varied for different RNAs and ranges from a low of 27% to high of 70%, with a mean value of 49%. The correspondence improves to a mean value of 81% when the analysis is limited to well-determined helices. In addition to well-determined helices, large well-determined structural domains can be observed in "energy dot plots" of some 16S ribosomal RNAs. The predicted domains correspond closely with structural domains that are found by the comparative method in the same RNAs. Our analyses also show that measuring the agreement between predicted and comparative secondary structure models underestimates the reliability of structural prediction by mfold.

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.

Testing the exon theory of genes: the evidence from protein structure.

A tendency for exons to correspond to discrete units of protein structure in protein-coding genes of ancient origin would provide clear evidence in favor of the exon theory of genes, which proposes that split genes arose not by insertion of introns into unsplit genes, but from combinations of primordial mini-genes (exons) separated by spacers (introns). Although putative examples of such correspondence have strongly influenced previous debate on the origin of introns, a general correspondence has not been rigorously proved. Objective methods for detecting correspondences were developed and applied to four examples that have been cited previously as evidence of the exon theory of genes. No significant correspondence between exons and units of protein structure was detected, suggesting that the putative correspondence does not exist and that the exon theory of genes is untenable.

Measuring residue associations in protein structures. Possible implications for protein folding.

We propose a number of distance measures between residues in protein structures based on average, minimum and maximum distances of all atom (backbone and side-chain) coordinates or with respect to side-chain atom coordinates only. The d1-distance (D1-distance) refers to the average distance between side-chain (backbone and side-chain) atoms of a residue pair in a given structure. The dm-distance (Dm-distance) refers to the minimum distance between side-chain atoms (non-trivial minimum distance between all atoms of a residue pair). For each distance measure, averaging and normalizing over representative protein structures, association values and closeness orderings for all amino acid types are determined. The expected associations of side-chain interactions between oppositely charged residues, among hydrophobic residues and of cysteine with cysteine are confirmed. Several surprising associations are observed relative to (1) the aromatic residues tyrosine and tryptophan, but not phenylalanine; (2) multiple histidine residues; (3) asymmetries of arginine versus lysine, aspartate versus glutamate, alanine versus glycine, and asparagine versus glutamine; (4) absence of correlations of alpha-carbon distances with side-chain distances. The all atoms D1-distance attractions are dominated by steric relationships, with glycine and alanine significantly close to all amino acids, whereas large residues are under-associated with all residue types. In contrast, for the closeness ordering corresponding to the minimum side-chain dm-distance, glycine and alanine are among the least associated. However, in the d1-distance alanine is significantly close to all hydrophobic residues with the exception of tryptophan. The dm-distance preferences display a pervasive attraction for tyrosine by almost all residue types, the prominence of tyrosine and tryptophan in cation-aromatic interactions, and the versatility of histidine in functionality. The principal findings suggest a new perspective on the early and intermediate stages of protein folding.

Structural analysis by energy dot plot of a large mRNA.

We have predicted the secondary structure of the entire 4217 nucleotide sequence of the genomic RNA of coliphage Q beta in one computer run using the computer program MFOLD that computes RNA structures within any prescribed increment of the computed minimum free energy. The results are presented in the form of an "energy dot plot" that shows both an optimal folding as well as the superposition of all base-pairs that can form in slightly suboptimal foldings. The plot reveals five large, well-determined, independent structural domains that cover approximately 50% of the viral genome. The predicted structural domains are consistent with and provide support for five large structural domains identified previously by quantitative electron microscopy in Q beta RNA. The dot plot also contains cluttered regions that indicate large numbers of alternative foldings within or between segments of an RNA molecule. These reflect the impossibility of accurate structure prediction and/or the biological reality of more than one folding. Weaker, long range structures, that are observed by electron microscopy in two alternate competing conformations, are located in the regions of the Q beta sequence that correspond to cluttered regions of the dot plot. The potential biological significance of these secondary structures is discussed.

Suboptimal sequence alignment in molecular biology. Alignment with error analysis.

A molecular sequence alignment algorithm based on dynamic programming has been extended to allow the computation of all pairs of residues that can be part of optimal and suboptimal sequence alignments. The uncertainties inherent in sequence alignment can be displayed using a new form of dot plot. The method allows the qualitative assessment of whether or not two sequences are related, and can reveal what parts of the alignment are better determined than others. It also permits the computation of representative optimal and suboptimal alignments. The relation between alignment reliability and alignment parameters is discussed. Other applications are to cyclical permutations of sequences and the detection of self-similarity. An application to multiple sequence alignment is noted.

A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison.

This article describes the latest version of an RNA folding algorithm that predicts both optimal and suboptimal solutions based on free energy minimization. A number of RNA's with known structures deduced from comparative sequence analysis are folded to test program performance. The group of solutions obtained for each molecule is analysed to determine how many of the known helixes occur in the optimal solution and in the best suboptimal solution. In most cases, a structure about 80% correct is found with a free energy within 2% of the predicted lowest free energy structure.

Predicting common foldings of homologous RNAs.

A new approach is proposed for determining common RNA secondary structures within a set of homologous RNAs. The approach is a combination of phylogenetic and thermodynamic methods which is based on the prediction of optimal and suboptimal secondary structures, topological similarity searches and phylogenetic comparative analysis. The optimal and suboptimal RNA secondary structures are predicted by energy minimization. Structural comparison of the predicted RNA secondary structures is used to find conserved structures that are topologically similar in all these homologous RNAs. The validity of the conserved structural elements found is then checked by phylogenetic comparison of the sequences. This procedure is used to predict common structures of ribonuclease P (RNAase P) RNAs.