On finding all suboptimal foldings of an RNA molecule.

An algorithm and a computer program have been prepared for determining RNA secondary structures within any prescribed increment of the computed global minimum free energy. The mathematical problem of determining how well defined a minimum energy folding is can now be solved. All predicted base pairs that can participate in suboptimal structures may be displayed and analyzed graphically. Representative suboptimal foldings are generated by selecting these base pairs one at a time and computing the best foldings that contain them. A distance criterion that ensures that no two structures are "too close" is used to avoid multiple generation of similar structures. Thermodynamic parameters, including free-energy increments for single-base stacking at the ends of helices and for terminal mismatched pairs in interior and hairpin loops, are incorporated into the underlying folding model of the above algorithm.

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.

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.

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.

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.

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.

Common structures of the 5' non-coding RNA in enteroviruses and rhinoviruses. Thermodynamical stability and statistical significance.

A total of 4051 suboptimal secondary structures are predicted by folding the 5' non-coding region of ten polioviruses, five human rhinoviruses and three coxsackieviruses using our new suboptimal folding algorithm for the prediction of both optimal and suboptimal RNA secondary structures. A comparative analysis of these RNA secondary structures reveals the conservation of common secondary structure that can be supported by phylogenetic data. The thermodynamic stability and statistical significance of these predicted, conserved helical elements are assessed and significant structure motifs in the 5' non-coding region are proposed. The possible roles of these structure motifs in the virus life cycle are discussed.

Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling.

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.

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.

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.

A new vitamin E analogue more active than alpha-tocopherol in the rat curative myopathy bioassay.

Vitamin E owes its biological effects to its antioxidant activity. Kinetic and mechanistic studies on phenolic antioxidants in vitro have led us to design and synthesize all-rac-2,4,6,7-tetramethyl-2-(4',8',12'-trimethyltridecyl) -5-hydroxy-3,4-dihydrobenzofuran, 3. In the rat curative myopathy bioassay the acetate of this compound has 1.5-1.9 times the bioactivity of all-rac-alpha-tocopherol acetate. This represents the first time that a rationally designed synthetic 'vitamin' has been found to have more activity in vivo than the corresponding natural vitamin.

The primary structure of the ribosomal A-protein (L12) from the halophilic eubacterium Haloanaerobium praevalens.

The ribosomal A-protein, equivalent to the ribosomal protein L12 from Escherichia coli, has been sequenced from the anaerobic halophilic eubacterium Haloanaerobium praevalens (DSM 2228). The protein contains 122 amino acids, has a composition of Asp6, Asn2, Thr2, Ser6, Glu22, Pro2, Gly13, Ala19, Val12, Met4, Ile5, Leu11, Phe3, Lys14, Arg1 and has a molecular weight of 12,691. The hydrophilicity profile was determined for this protein. A phylogenetic or cluster tree was calculated from computer analysis of the sequence data on eubacterial ribosomal A-proteins. H. praevalens clusters with a group that includes Bacillus subtilis, Micrococcus lysodeikticus, Bacillus stearothermophilus and Clostridium pasteurianum.

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.

Equivalency of linear least squares curve fitting and reciprocal functions in protein circular dichroic spectra analysis.

Two methods for the analysis of the circular dichroism spectra of proteins for determination of secondary structure have been examined. These are the linear curve fitting of the data, minimized in the least squares sense, and the method of reciprocal functions proposed by C.C. Baker and I. Isenberg, Biochemistry 15 (1976) 629. It is shown that the use of these two methods give results that are identical, providing the same set of reference spectra are used in each case, and, therefore, that no new information is obtained by the use of either one over the other.

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.

The measurement of rectal and testis temperature in conscious mice, with observations on the effect of direct heating.

The application of Newton's law of cooling to freshly killed mice was found not to measure accurately their rectal or testis temperature. Improvements in the fitting process gave satisfactory results for rectal temperature only. A diffusion model was applied to testis cooling but was of no avail. Finally a satisfactory correction factor was determined empirically. This method was applied to conscious mice whose hindquarters were immersed in a stirred oilbath at 34 to 42 degrees C for 1 h, and to controls. It was found that both rectal and testis temperatures increased with bath temperature, producing a graph with a slope of only 0.5, indicating a regulatory capacity. Conscious mice, but not anaesthetised, can maintain a testis temperature of 39 degrees C in a bath at 42 degrees C.

[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.