Oscillation frequencies of tapered plant stems

Free oscillations of upright plant stems, or in technical terms, slender tapered rods with one end free, can be described by considering the equilibrium between bending moments in the form of a differential equation with appropriate boundary conditions. For stems with apical loads, where the mass of the stem is negligible, Mathematica 4.0 returns solutions for tapering modes α = 0, 0.5, and 1. For other values of α, including cases where the modulus of elasticity varies over the length of the stem, approximations leading to an upper and a lower estimate of the frequency of oscillation can be derived. For the limiting case of ω = 0, the differential equation is identical with Greenhill's equation for the stability against Euler buckling of a top-loaded slender pole. For stems without top loads, Mathematica 4.0 returns solutions only for two limiting cases, zero gravity (realized approximately for oscillations in a horizontal orientation of the stem) and for ω = 0 (Greenhill's equation). Approximations can be derived for all other cases. As an example, the oscillation of an Arundo donax plant stem is described.     

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.     

Role of the ITS2-proximal stem and evidence for indirect recognition of processing sites in pre-rRNA processing in yeast

Eucaryotic ribosome biogenesis involves many cis-acting sequences and trans-acting factors, including snoRNAs. We have used directed mutagenesis of rDNA plasmids in yeast to identify critical sequence and structural elements within and flanking the ITS2-proximal stem. This base paired structure, present in the mature ribosome, is formed between the 5′-end of 25S and the 3′-end of 5.8S rRNAs. Previously we demonstrated that formation of this structure was critical for pre-rRNA processing in yeast. Here we show that there are no sequence-specific recognition elements within the ITS2-proximal stem, rather the structure of this stem is critical for processing. This stem cannot exceed a specific length, but there are different length restrictions for different regions within this tripartite stem. Neither the conserved unpaired nucleotides within the stem nor the sequence of the mature rRNA at the processing sites are required for processing. Collectively, these results suggest a measuring model whereby initial cleavage within ITS2 at the C2 processing site and termination of subsequent exonuclease activity yielding the mature termini are affected by the relative position of sequence and structural elements within the ITS2-proximal stem.     

Water, Shape Recognition, Salt Bridges, and Cation-Pi Interactions Differentiate Peptide Recognition of the HIV Rev-Responsive Element

Recognition of the human immunodeficiency virus Rev-responsive element (RRE) RNA by the Rev protein is an essential step in the viral life cycle. Formation of the Rev-RRE complex signals nucleocytoplasmic export of unspliced and partially spliced viral RNA. Essential components of the complex have been localized to a minimal arginine-rich Rev peptide and stem IIB of RRE. In vitro selection studies have identified a synthetic peptide known as RSG 1.2 that binds with better specificity and affinity to RRE than the Rev peptide. NMR structures of both peptide-RNA complexes of Rev and RSG 1.2 bound to RRE stem IIB have been solved and reveal gross structural differences between the two bound complexes. Molecular dynamics simulations of the Rev and RSG 1.2 peptides in complex with RRE stem IIB have been simulated to better understand on an atomic level how two arginine-rich peptides of similar length recognize the same sequence of RNA with such different structural motifs. While the Rev peptide employs some base-specific hydrogen bonding for recognition of RRE, shape recognition, through contact with the sugar-phosphate backbone, and cation-pi interactions are also important. Molecular dynamics simulations suggest that RSG 1.2 binds more tightly to the RRE sequence than Rev by forming more base-specific contacts, using water to mediate peptide-RNA contacts, and is held in place by a strong salt bridge network spanning the major groove of the RNA.     

Exploring the complex folding kinetics of RNA hairpins: II. Effect of sequence, length, and misfolded states

The complexity of RNA hairpin folding arises from the interplay between the loop formation, the disruption of the slow-breaking misfolded states, and the formation of the slow-forming native base stacks. We investigate the general physical mechanism for the dependence of the RNA hairpin folding kinetics on the sequence and the length of the hairpin loop and the helix stem. For example, 1), the folding would slow down when a stable GC basepair moves to the middle of the stem; 2), hairpin with GC basepair near the loop would fold/unfold faster than the one with GC near the tail of the stem; 3), within a certain range of the stem length, a longer stem can cause faster folding; and 4), certain misfolded states can assist folding through the formation of scaffold structures to lower the entropic barrier for the folding. All our findings are directly applicable and quantitatively testable in experiments. In addition, our results can be useful for molecular design to achieve desirable fast/slow-folding hairpins, hairpins with/without specific misfolded intermediates, and hairpins that fold along designed pathways.     

WSE, a new sequence distance measure based on word frequencies

In this article, we present a new distance metric, the Weighted Sequence Entropy (WSE), based on the short word composition of biological sequences. As a revision of the classical relative entropy (RE), our metric (1) works equivalently with RE in the case of small k, (2) avoids the degeneracy when some word types are absent in one sequence but not in the other. Experiments on 25 viruses including SARS-CoVs show that our method and RE give exactly the same phylogenetic tree when word length k3. When k>3, our method still works and gets convergent phylogenetic topology but the RE gives degenerate results.     

An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA.

The RNA polymerase gene (gene 1) of the human coronavirus 229E is approximately 20 kb in length and is located at the 5' end of the positive-strand genomic RNA. The coding sequence of gene 1 is divided into two large open reading frames, ORF1a and ORF1b, that overlap by 43 nucleotides. In the region of the ORF1a/ORF1b overlap, the genomic RNA displays two elements that are known to mediate (-1) ribosomal frameshifting. These are the slippery sequence, UUUAAAC, and a 3' pseudoknot structure. By introducing site-specific mutations into synthetic mRNAs, we have analysed the predicted structure of the HCV 229E pseudoknot and shown that besides the well-known stem structures, S1 and S2, a third stem structure, S3, is required for a high frequency of frameshifting. The requirement for an S3 stem is independent of the length of loop 2.     

Analysis of the Primary Sequence and Secondary Structure of the Unusually Long SSU rRNA of the Soil Bug, Armadillidium vulgare

The complete nucleotide sequence of the SSU rRNA gene from the soil bug, Armadillidium vulgare (Crustacea, Isopoda), was determined. It is 3214 bp long, with a GC content of 56.3%. It is not only the longest SSU rRNA gene among Crustacea but also longer than any other SSU rRNA gene except that of the strepsipteran insect, Xenos vesparum (3316 bp). The unusually long sequence of this species is explained by the long sequences of variable regions V4 and V7, which make up more than half of the total length. RT-PCR analysis of these two regions showed that the long sequences also exist in the mature rRNA and sequence simplicity analysis revealed the presence of slippage motifs in these two regions. The putative secondary structure of the rRNA is typical for eukaryotes except for the length and shape variations of the V2, V4, V7, and V9 regions. Each of the V2, V4, and V7 regions was elongated, while the V9 region was shortened. In V2, two bulges, located between helix 8 and helix 9 and between helix 9 and helix 10, were elongated. In V4, stem E23-3 was dramatically expanded, with several small branched stems. In V7, stem 43 was branched and expanded. Comparisons with the unusually long SSU rRNAs of other organisms imply that the increase in total length of SSU rRNA is due mainly to expansion in the V4 and V7 regions. Received: 2 March 1999 / Accepted: 22 July 1999     

Rhythmic variations in purine run frequencies in bacteriophage RNA

The RNA of small bacteriophages is known to exhibit rhythmic deviations from random in the frequencies of runs of purine bases, with runs of even length falling short of expected frequency and those of odd length being in excess. The detailed positions and frequencies of these runs are examined in the case of bacteriophage MS2, for which the complete RNA sequence has been determined, and comparisons are made with other RNA sequences. It is found that a strong rhythmic effect is localized in two subregions of the MS2 genome, in part of the RNA of phage Qβ and in three RNAs transcribed from phage λ. The statistical significance of the deviations from random is assessed, and hypotheses about the origin, evolution and maintenance of the effect are considered.     

Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA

Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.     

油菜叶绿体16SrRNA基因的一级结构分析

本文报道了油菜叶绿体16S rRNA基因的全顺序及其5′端上游的156bp和3′端下游的101bp的核苷酸顺序。油菜叶绿体16s rRNA基因长为1491bp,和烟草、玉米相比,同源程度分别为98.5％、96.1％。油菜叶绿体16S rRNA基因5′端上游及3′端下游的顺序能互补而形成一个较大的茎环结构,但与烟草相比,由于3′端下游顺序有79bp的缺失,因此,该结构中的茎部分大小仅为烟草的二分之一。     

Estimation of allele frequencies for VNTR loci

VNTR loci provide valuable information for a number of fields of study involving human genetics, ranging from forensics (DNA fingerprinting and paternity testing) to linkage analysis and population genetics. Alleles of a VNTR locus are simply fragments obtained from a particular portion of the DNA molecule and are defined in terms of their length. The essential element of a VNTR fragment is the repeat, which is a short sequence of basepairs. The core of the fragment is composed of a variable number of identical repeats that are linked in tandem. A sample of fragments from a population of individuals exhibits substantial variation in length because of variation in the number of repeats. Each distinct fragment length defines an allele, but any given fragment is measured with error. Therefore the observed distribution of fragment lengths is not discrete but is continuous, and determination of distinct allele classes is not straightforward. A mixture model is the natural statistical method for estimating the allele frequencies of VNTR loci. In this article we develop nonparametric methods for obtaining the distribution of allele sizes and estimates of their frequencies. Methods for obtaining maximum-likelihood estimates are developed. In addition, we suggest an empirical Bayes method to improve the maximum-likelihood estimates of the gene frequencies; the empirical Bayes procedure effects a local smoothing. The latter method works particularly well when measurement error is large relative to the repeat size, because the estimated distribution of allele frequencies when maximum likelihood is used is unreliable because of an alternating pattern of over- and underestimation. We define alleles and estimate the allele frequencies for two VNTR loci from the human genome (D17S79 and D2S44), from data obtained from Lifecodes, Inc.     

Functions of the stem region of the Semliki Forest virus fusion protein during virus fusion and assembly

Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.     

Structure-function relationships of shared-stem and conventional molecular beacons

Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.     

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.     

GC content dependency of open reading frame prediction via stop codon frequencies

A frequently used approach for detecting potential coding regions is to search for stop codons. In the standard genetic code 3 out of 64 trinucleotides are stop codons. Hence, in random or non-coding DNA one can expect every 21st trinucleotide to have the same sequence as a stop codon. In contrast, the open reading frames (ORFs) of most protein-coding genes are considerably longer. Thus, the stop codon frequency in coding sequences deviates from the background frequency of the corresponding trinucleotides. This has been utilized for gene prediction, in particular, in detecting protein-coding ORFs. Traditional methods based on stop codon frequency are based on the assumption that the GC content is about 50%. However, many genomes show significant deviations from that value. With the presented method we can describe the effects of GC content on the selection of appropriate length thresholds of potentially coding ORFs. Conversely, for a given length threshold, we can calculate the probability of observing it in a random sequence. Thus, we can derive the maximum GC content for which ORF length is practicable as a feature for gene prediction methods and the resulting false positive rates. A rough estimate for an upper limit is a GC content of 80%. This estimate can be made more precise by including further parameters and by taking into account start codons as well. We demonstrate the feasibility of this method by applying it to the genomes of the bacteria Rickettsia prowazekii, Escherichia coli and Caulobacter crescentus, exemplifying the effect of GC content variations according to our predictions. We have adapted the method for predicting coding ORFs by stop codon frequency to the case of GC contents different from 50%. Usually, several methods for gene finding need to be combined. Thus, our results concern a specific part within a package of methods. Interestingly, for genomes with low GC content such as that of R. prowazekii, the presented method provides remarkably good results even when applied alone.     

Substrate recognition by human RNase P: identification of small, model substrates for the enzyme.

RNase P from HeLa cells can efficiently cleave tRNA precursor molecules in vitro but cannot cleave potential substrates from which the D, anticodon and variable loops and stems of the tRNA moiety have all been removed. However, molecules from which the latter subdomains have been removed individually do serve as substrates. We show here that molecules that contain only a 5' leader sequence, the acceptor stem and the T stem and loop of the tRNA domain, and a bulge as small as one nucleotide downstream from nucleotide 7 in the tRNA sequence at the junction of the two stems, can serve as substrates for human RNase P. The identity of the nucleotide in the bulge is important in determining both the efficiency of the cleavage and the conformation of the substrate and/or the enzyme-substrate complex. We also show that the human enzyme locates the appropriate site for cleavage of its substrates in part by 'measuring' the length of the helices in the acceptor and T stems in both model and natural substrates.     

Different repetition frequencies of a 120 base-pair DNA-element and its arrangement in Chironomus thummi thummi and Chironomus thummi piger

The repetition frequency of a highly repetitive DNA sequence has been measured in the genomes of Ch. thummi thummi and Ch. th. piger. This sequence is known to be involved in the evolutionary duplication of defined chromosomal segments leading to a significant increase in the genome size of Ch. th. thummi. Reassociation of this highly repetitive DNA sequence which has a repeat length of 120 base-pairs, with total Ch. th. thummi and Ch. th. piger DNA has shown that the repetition frequency in the Ch. th. thummi DNA is 5.5 fold higher than in Ch. th. piger. In both genomes a 120 base-pair sequence is present as tandemly repeated sequence as shown by Southern analysis.