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PubMed1984

Theoretical and experimental approach to recognition of amino acid by tRNA and nucleotide II.

Strong and specific interactions between amino acids and their cognate tRNAs have been found by the CD, fluorescence, and UV absorption difference spectroscopy and by the gel-filtration method. New types of strong hydrogen bonding interactions between an amino acid and polynucleotides, nucleotides, nucleosides, and nucleic acid bases in aqueous solution have also been detected. These findings are strong experimental evidence for the C4N model of the genetic code. The cases of glutamate and aspartate will theoretically be discussed in details.

Amino Acids↗

PubMed1998

Sequences of ten circular ssDNA components associated with the milk vetch dwarf virus genome.

Milk vetch dwarf virus (MDV) is a member of the proposed genus Nanovirus, and its genome is composed of multiple, circular ssDNA components of about 1 kb. We have cloned and sequenced ten ssDNA components and designated them MDV-C1 to C10. Each DNA component contains one potential major open reading frame, and contains a putative stem-loop structure in the non-coding region. Notably, four components (C1, C2, C3 and C10) encode distinct replication-associated (Rep) proteins of 33 kDa, which show only limited (42-57%) amino acid identity. The six other components encode proteins with calculated molecular masses ranging from 12.7 to 19.7 kDa. Comparison of the sequences with those of other nanoviruses reveals that MDV is closely related to faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus (SCSV). Six putative MDV genome products, including one Rep and five non-Rep proteins, show high (70.4-90.9%) amino acid identity to the corresponding six FBNYV proteins, whereas two other Rep proteins encoded by MDV-C2 and C3 are 82.3% and 73.0% identical to those encoded by SCSV-C2 and C6, respectively. These results indicate that MDV, FBNYV and SCSV have diverged from a common origin, which had multiple Rep components. In addition, the putative proteins encoded by MDV-C4 and its homologues contain a consensus retinoblastoma-binding motif, suggesting that they may be involved in controlling the host cell cycle.

Amino Acid Sequence↗

PubMed1985

Introns, protein syntheses and aging.

In the fungus Podospora, a correlation has recently been established between the presence of circular DNA molecules arising from the mitochondrial genome (SEN-DNAs) and the senescence syndrome. Here, I propose a hypothesis which accounts for the initial event which leads to the first SEN-DNA. A molecule in the most frequent situation where the SEN-DNA is an intron which might code for a maturase. This hypothesis is based upon several observations made either in Podospora or in the yeast S. cerevisiae. It assumes that mitochondrially synthesized maturases are unspecific nucleases able to work at the level of RNA and DNA molecules. Their specificity for RNA splicing instead of DNA is given by cytoplasmic proteins. Therefore, if the balance between cytoplasmic and mitochondrial protein syntheses is disturbed in favour of the mitochondrial compartment, the maturase would be accumulated and allowed to splice introns from DNA instead of RNA molecules. This hypothesis can account for aging of higher eucaryotic cells by postulating analogous processes in their nuclear compartment.

Ascomycota↗

PubMed1995

Secondary rearrangements and post-rearrangement selection contribute to restricted immunoglobulin DJH expression in young rabbit bone marrow.

Extrachromosomal circular DNA purified from bone marrow cells of 2-weeks-old rabbits was assayed by polymerase chain reaction to determine the relative rearrangement frequencies of immunoglobulin DH to JH genes in vivo. DH genes rearranged to individual JH genes with different frequencies. This bias did not correlate with potential sequence overlaps in the DH or JH coding sequences. The JH2 and JH4 genes were the preferred targets of recombination in primary rearrangements. Although primary rearrangements to JH6 were relatively infrequent, secondary rearrangements were detected. This assay also revealed previously undescribed JH pseudogenes with functional recombination signal sequences. Analyses of genomic VDJH indicated that B cells expressing VDJH4 heavy chains survived and dominated in the bone marrow environment due to secondary rearrangements and/or post-rearrangement selection.

Amino Acid Sequence↗

PubMed2003

Direct numerical simulation of the motion of circular pollutant particles in Newtonian fluid.

An improved implementation of distributed multiplier/fictitious domain method is presented for the direct numerical simulation of particulate flow. The key improvement is to replace a finite-element triangulation for the velocity and a "twice-coarser" triangulation for the pressure with a rectangular discretization for the velocity and pressure. For code validation, the sedimentation of a single particle in a two-dimensional channel was simulated. The results showed that the simulation is independent of the mesh size as well as the time step. The comparison between experimental data and this simulation showed that our code can give a more accurate simulation on the motion of particles than previous DLM code. The code was then applied to simulate the sedimentation of 600 particles in a rectangular box. The falling course is presented and discussed. At the same time, this simulation also demonstrates that the method presented in this paper can be used for solving the initial problems involving a lager number of particles exactly with computing durations kept at acceptable levels.

Algorithms↗

PubMed1999

Structural studies on a 2,3-diphosphoglycerate independent phosphoglycerate mutase from Bacillus stearothermophilus.

Phosphoglycerate mutase (PGM), an important enzyme in the glycolytic pathway, catalyzes the transfer of a phosphate group between the 2 and the 3 positions of glyceric acid. The gene coding for the 2, 3-diphosphoglycerate independent monomeric PGM from Bacillus stearothermophilus (57 kDa), whose activity is extremely pH sensitive and has an absolute and specific requirement for Mn2+, has been cloned and the enzyme overexpressed and purified to homogeneity. Circular dichroism studies showed at most only small secondary structure changes in the enzyme upon binding to Mn2+ or its 3-phosphoglycerate substrate, but thermal unfolding analyses revealed that Mn2+ but not 3-phosphoglycerate caused a large increase in the enzyme's stability. Diffraction-quality crystals of the enzyme were obtained at neutral pH in the presence of 3-phosphoglyceric acid with ammonium sulfate as the precipitating agent; these crystals diffract X rays to beyond 2.5-A resolution and belong to the orthorhombic space group C2221 with unit cell dimensions, a = 58.42, b = 206.08, c = 124.87 A, and alpha = beta = gamma = 90.0 degrees. The selenomethionyl version of the B. stearothermophilus protein has also been overexpressed, purified, and crystallized. Employing these crystals, the determination of the three-dimensional structure of this PGM by the multiwavelength anomalous dispersion method is in progress.

2,3-Diphosphoglycerate↗

PubMed2003

The theta-defensin, retrocyclin, inhibits HIV-1 entry.

Retrocyclin is a circular antimicrobial 18-residue peptide encoded in the human genome by a theta-defensin pseudogene. In the human genome, the gene for retrocyclin is inactivated by an in-frame stop codon in its signal sequence but its mature coding sequence is intact. The peptide corresponding to the processed human retrocyclin, generated by solid phase peptide synthesis, inhibited replication of R5 and X4 strains of HIV-1 in human cells. Luciferase reporter virus and Vpr-BLaM entry assays were used to demonstrate that retrocyclin specifically blocked R5 and X4 HIV-1 replication at entry. Surface plasmon resonance demonstrated that retrocyclin bound to soluble CD4 and gp120, but gp120 cell-binding assays revealed that retrocyclin did not fully inhibit the binding of soluble CD4 to gp120. A fluorescent retrocyclin congener localized in cell-surface patches either alone or colocalized with CD4, CXCR4, and CCR5. In the aggregate, these results suggest that retrocyclin blocks an entry step in HIV-1 replication. Retrocyclin represents a new class of small molecule HIV-1 entry inhibitors.

Anti-HIV Agents↗

PubMed1989

Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.

Base Sequence↗

PubMed1999

Linear SRY transcript in equine testis.

Employing a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques, the complete coding sequence of cDNA for the equine SRY gene was determined. We also attempted to make clear whether the equine SRY gene transcript is expressed in the adult testis, and whether the type of transcript is expressed as linear or circular RNA. As a result, in total a 1420 bp cDNA sequence was determined. Accomplishment of 3' RACE infers that equine SRY gene was expressed as a linear RNA transcript in testicular tissue just after puberty, in contrast to the situation in mice.

Amino Acid Sequence↗

PubMed2025

Suppression of errors in collectively coded information.

Modern life largely transmits genetic information from mother to daughter through the duplication of single physically intact molecules that encode information. However, copying an extended molecule requires complex copying machinery and high fidelity that scales with the genome size to avoid the error catastrophe. Here, we explore these fidelity requirements in an alternative architecture, the virtual circular genome, in which no one physical molecule encodes the full genetic information. Instead, information is encoded and transmitted in a collective of overlapping and interacting segments. Using a model experimental system of a complex mixture of DNA oligomers that can partly anneal and extend off each other, we find that mutant oligomers are suppressed relative to a model without collective encoding. Through simulations and theory, we show that this suppression of mutants can be explained by competition for productive binding partners. As a consequence, information can be propagated robustly in a virtual circular genome even at mutation rates expected under prebiotic conditions.

Journal Article↗

PubMed1997

An alpha-helical signal in the cytosolic domain of the interleukin 2 receptor beta chain mediates sorting towards degradation after endocytosis.

High-affinity IL2 receptors consist of three components, the alpha, beta, and gamma chains that are associated in a noncovalent manner. Both the beta and gamma chains belong to the cytokine receptor superfamily. Interleukin 2 (IL2) binds to high-affinity receptors on the cell surface and IL2-receptor complexes are internalized. After endocytosis, the components of this multimolecular receptor have different intracellular fates: one of the chains, alpha, recycles to the plasma membrane, while the others, beta and gamma, are routed towards late endocytic compartments and are degraded. We show here that the cytosolic domain of the beta chain contains a 10-amino acid sequence which codes for a sorting signal. When transferred to a normally recycling receptor, this sequence diverts it from recycling. The structure of a 17-amino acid segment of the beta chain including this sequence has been studied by nuclear magnetic resonance and circular dichroism spectroscopy, which revealed that the 10 amino acids corresponding to the sorting signal form an amphipathic alpha helix. This work thus describes a novel, highly structured signal, which is sufficient for sorting towards degradation compartments after endocytosis.

Amino Acid Sequence↗

PubMed1987

Site-specific recombination directed by single-stranded crossover linkers: specific deletion of the amino-terminal region of the beta-galactosidase gene in pUC plasmids.

The "duplex crossover linker" technique was simplified and used to delete the beta-galactosidase (beta-Gal)-coding sequence upstream from the multiple restriction sites in pUC plasmids. A single-stranded crossover linker, with a homology-searching sequence as short as 5 bases, was initially ligated to a linearized plasmid. Inside Escherichia coli, the plasmid was circularized by intramolecular, homologous recombination between the (5'-or 3'-) protruding homology-searching sequence and a targeted region in the opposite terminus. As a consequence, sequences beyond the point of integration were deleted. Specific deletion of sequences up to 1472 bp was demonstrated. The single-stranded linkers apparently avoided generation of undesirable mutants associated with the usage of duplex linkers. A mechanism has been proposed for the intramolecular recombination directed by the crossover linkers. It principally involves either 3'- or 5'-exonucleolytic breakdown of the homologous terminus of the plasmid, circularization by spontaneous pairing of the exposed complementary strands, and subsequent degradation of any redundant sequence.

Base Sequence↗

PubMed2026

The first complete mitochondrial genome of Strigea falconis (Digenea: Strigeidae) reveals six tandemly repeated trnE-containing units and provides mt evidence for the non-monophyly of the family Strigeidae.

BACKGROUND: Phylogenetic relationships among members in the order Diplostomida remain contentious, with mitochondrial (mt) and nuclear genomic data often yielding conflicting topologies. A major limitation is the availability of only a few mt genomes from the type genus Strigea, hindering a robust test of the monophyly of the family Strigeidae and the order Diplostomida. RESULTS: The mt genome of S. falconis was completely sequenced for the first time, which was a circular molecule of 16,872 bp in length, encoding the typical set of 36 mt genes and six duplicate tRNA-Glu genes. Notably, there were seven identical and consecutive tandem repeat units each consist of a 169 bp non-coding region followed by a trnE gene in the newly assembled genome. Phylogenomic analyses based on concatenated predicted amino acid sequences of 12 proteins robustly placed S. falconis in the same clade as Apharyngostrigea pipientis. Crucially, the family Strigeidae was not recovered as monophyletic. Instead, two species within Strigeidae, Cardiocephaloides medioconiger and Cotylurus marcogliesei, clustered with representatives of Diplostomidae, providing mt evidence for the paraphyly of Strigeidae under the current sampling. CONCLUSIONS: The newly sequenced mt genome of S. falconis reveals a previously unreported six-copy tandem repeat of trnE-containing units among currently available diplostomoid mt genomes. Phylogenetic analyses based on mt protein-coding genes provide additional mt evidence that the family Strigeidae was not recovered as monophyletic under the present taxon sampling. However, because mt genomes represent a single maternally inherited linkage group, broader taxon sampling, independent nuclear phylogenomic data, and explicit sensitivity analyses will be required to confirm these relationships and guide any formal systematic revision.

Animals↗

PubMed2026

Comparative genomics and phylogenetic analysis of three Malvaceae species on the basis of chloroplast genomes.

INTRODUCTION: The Malvaceae family shows rich species diversity and has substantial economic and medicinal value. However, the frequent interspecific hybridization among members of this family has resulted in confused phylogenetic relationships among the groups, limiting the usefulness of traditional classification methods. METHODS: This study aimed to investigate the phylogenetic relationships among selected taxa of Malvaceae by evaluating 23 chloroplast (CP) genomes, including three newly assembled CP genomes. Among these three genomes, the CP genome of Hibiscus schizopetalus L. was reported for the first time, while the CP genomes of Alcea rosea L. and Hibiscus grewiifolius L., which have been deposited in NCBI, were re-analyzed here alongside newly generated data for comparative purposes. In addition, 20 downloaded CP genomes encompassing 13 genera were analyzed using SNPs in whole CP genomes data. RESULTS: The results showed that the genomes ranged from 160,403 to 161,978 base pairs in length and consisted of small single copies (SSCs) and large single copies (LSCs) separated by two inverted repeat sequences (IRs), forming a typical quadripartite circular structure. The entire genome sequence showed relative conservation across species in terms of structure, GC content, codon usage, and gene composition. The mutation sites were mainly located in the LSC and SSC regions, and the variability in the non-coding regions was higher than that in the coding regions. The nucleotide polymorphism (Pi) analysis identified the non-coding regions such as ndhF-rpl32 and psbZ-trnG as high variable hotspots. A maximum likelihood phylogenetic tree was constructed based on SNPs in whole CP genomes data. The phylogenetic analysis divided these 23 species into five highly supported clades. It also revealed a close sister-group relationship between Abelmoschus and Hibiscus species, suggesting that Hibiscus may have a separate lineage from okra species. DISCUSSION: In conclusion, the increasing availability of CP genome resources will enhance our understanding of the classification and evolutionary patterns of the Malvaceae family. The development of molecular markers will provide important molecular evidence for precise identification and classification revision of plants in this family.

Malvaceae↗

PubMed2004

On the peptide-antipeptide interactions in interleukin-1 receptor system.

Interleukin-1 receptor antagonist (IL-1Ra) and vaccinia virus protein C10L share a VTXFYF motif, with X being Lys or Arg residue, respectively. Peptides of such sequence compete successfully with IL-1 for the cellular receptor. A pair of complementary peptides, based on the Siemion's hypothesis on the periodicity of the genetic code (QWLNIN and QWANIN), and another pair, in which, following the Root- Bernstein theory, Lys was used as complementary amino acid to Phe (QWLKIK and QWAKIK), were investigated for the peptide-antipeptide interactions using mass spectrometry (ESI-MS) and circular dichroism (CD) methods. The CD measurements indicated some conformational changes, more pronounced in the Siemion's pairs, however, no heterodimer formation was found by MS. In the region of IL-1 receptor situated close to the position of IL-1Ra in the IL-1Ra-receptor complex, a KQKL motif is present, suggesting a possibility of complementary recognition of the Root-Bernstein type in the IL-1 receptor. The biological activity of the complementary peptides is similar to that of the original ones. They efficiently compete with IL-1 and show moderate immunosuppressory activity in humoral and cellular immune response. The inhibition of the IL-1-IL-1 receptor interaction may result from the complementary peptides acting as mini-receptors with affinity for IL-1.

Amino Acid Sequence↗

PubMed1999

MCM21 and MCM22, two novel genes of the yeast Saccharomyces cerevisiae are required for chromosome transmission.

The minichromosome maintenance genes, MCM21 and MCM22, have been cloned and are shown to code for the ORFs YDR318W and YJR135C respectively. Mutations in these genes caused a decrease in the stability of the minichromosome. This decrease in stability was associated with an increase in the copy number of the minichromosome in cells carrying it. Small circular dicentric plasmids were maintained relatively stably and structurally intact in the mutants compared with the wild-type strain. In the latter, such plasmids were mitotically unstable and, upon recovery, showed frequent rearrangements of their DNA. A centromere offered less obstruction to transcription in mutant cells than in the wild type, showing that both these mutants had a more relaxed kinetochore assembly. The mutant strains showed elevated rates of chromosome loss but not those of recombination. Both the mutations caused the cells to display a higher sensitivity towards the anti-mitotic drug benomyl. All these observations suggest that MCM21 and MCM22 are important for chromosome segregation with a potential role in kinetochore function. These genes are non-essential, as their deletions from chromosomes did not cause loss of cell viability. However, exponentially growing mutant cells carrying the deletion of the MCM21 gene had a significant population of large-budded cells with a single nucleus at the neck. Furthermore, the DNA content of these cells showed a shift towards 2N, suggesting a temporary pause of cells in G2 or in an early phase of mitosis. The mcm21 and mcm22 mutations do not show synthetic lethality or any further enhancement of growth defects, implying that they could be carrying out non-overlapping functions in chromosome segregation.

Cell Cycle Proteins↗

PubMed1999

The enteric nervous system: region and target specific projections and neurochemical codes.

The goal of this report is to summarise the current knowledge on the projection pathways of enteric neurones innervating the muscle and mucosa in different regions of the gut. Combination of neuronal tracing, immunohistochemical and electrophysiological methods has allowed researchers to gain insight into the enteric hardwiring of specific target tissue in the gut. A polarised innervation pattern of the circular muscle was demonstrated for the stomach fundus/corpus and the ileum with descending pathways being primarily nitrergic while ascending pathways were primarily cholinergic. This characteristic hardwiring is thought to set in part the functional basis for peristalsis. A similar polarised innervation pathway was found for the enteric innervation of the mucosa in the stomach and large intestine but not in the small intestine. In both the stomach (myenteric neurones) and in the proximal and distal colon (submucosal neurones), ascending pathways to the mucosa are primarily cholinergic while descending pathways are primarily non-cholinergic. In the colon, results suggest that activation of both pathways induces a cross potentiation of cholinergic and vasoactive intestinal polypeptidergic mediated secretion. Furthermore, a large population of myenteric neurone s projecting to the mucosa in the small and large intestine are probably intrinsic primary afferent neurones sensitive to mechanical as well as chemical stimuli.

Animals↗

PubMed1995

Expression of mouse uterine peptidylarginine deiminase in Escherichia coli: construction of expression plasmid and properties of the recombinant enzyme.

To study the structure/function relationships of peptidylarginine deiminase (PAD), we constructed an Escherichia coli expression plasmid for mouse uterine PAD. First, segments of a cDNA encoding murine PAD were subcloned into a single plasmid, and the resulting plasmid, pKSPAD1, was inserted into an expression vector, pKK223-3, at the EcoRI and HindIII restriction sites. Since no detectable amount or activity of the PAD was produced by E. coli carrying that plasmid, the 5'-untranslated sequence of the cDNA was replaced with several synthetic DNAs. One of the constructed plasmids, pKKPAD4, which had a unique DNA linker containing a pair of Shine-Dalgarno sequences and a short preceding cistron inserted into the adjacent 5'-region of the coding region, produced a large quantity of mouse PAD as an unfused protein in E. coli. The purified recombinant PAD was indistinguishable from the native enzyme with respect to some structural properties, such as molecular mass, amino- and carboxyl-terminal sequences, and circular dichroism spectra. However, the alpha-amino group of the amino-terminal methionine residue of the recombinant PAD was not acetylated as was that of the native enzyme. Comparison of the recombinant PAD with the natural enzyme did not indicate significant differences in their sensitivity to activation by Ca2+ and in their substrate specificity toward arginine derivatives. The rates of modification of soybean trypsin inhibitor (Kunitz) were also similar for the recombinant and native PADs. These results indicate that the recombinant PAD has biological activities identical to those of the native enzyme and that the N alpha-acetyl group in the native PAD does not appear to have any particular role in the enzyme's catalytic function.

Amino Acid Sequence↗