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At least 271 records · Page 15Linked to original sources
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↗
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↗
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↗
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↗
PubMed1984

Simian virus 40 T antigen is required for viral excision from chromosomes.

We describe experiments that show that simian virus 40 (SV40) T antigen is required for viral excision from host chromosomes at some point prior to or during the homologous recombination events that create circular wild-type virus. Two recombinant SV40-pBR322 plasmids were constructed such that homologous recombination across similar-sized but different duplications of SV40 would reconstitute wild-type viral DNA. One plasmid (pSVED) was constructed such that the duplication separates the viral early T-antigen promoter from the coding sequences; the other recombinant (pSVLD) contains a duplication of the late viral sequences and thus maintains a complete T-antigen gene. These plasmids were individually established in Rat 2 cells via cotransformation with the herpes virus Tk gene. Both classes of cell lines contained integrated tandem arrays of the plasmids and yielded equivalent levels of infectious virus after cell fusions with COS-7 cells; however, only the T+ lines yielded virus after cell fusion with CV-1 cells. These results are consistent with the notion that viral excision is initiated by T-antigen-mediated in situ replication of viral DNA as proposed in the "onion skin" model. In contrast, both plasmids yielded infectious virus when transiently introduced via transfection into CV-1 cells. This latter finding is discussed in terms of the possible induction of cellular repair and recombination pathways evoked by the introduction of damaged DNA into the nucleus.

Animals↗
PubMed1972

Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli.

We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.

Centrifugation, Density Gradient↗
PubMed1978

BK virus DNA: cleavage map and sequence analysis.

A detailed physical map of the BK virus (MM strain) genome has been constructed with respect to the cleavage sites of 11 different restriction enzymes. The enzymes cut BKV(MM) DNA at 61 specific sites whose locations have been determined. Preliminary nucleotide sequence was carried out in the region from 0.70-0.75 map positions on BKV(MM) DNA. An 80% homology was found at 0.714-0.744 map positions on BKV(MM) DNA with 0.722-0.752 map positions on simian virus 40 DNA. This region of simian virus 40 DNA codes for the synthesis of the leader sequence of late mRNA.

Animals↗
PubMed1992

Molecular code for cooperativity in hemoglobin.

Although tetrameric hemoglobin has been studied extensively as a prototype for understanding mechanisms of allosteric regulation, the functional and structural properties of its eight intermediate ligation forms have remained elusive. Recent experiments on the energetics of cooperativity of these intermediates, along with assignments of their quaternary structures, have revealed that the allosteric mechanism is controlled by a previously unrecognized symmetry feature: quaternary switching from form T to form R occurs whenever heme-site binding creates a tetramer with at least one ligated subunit on each dimeric half-molecule. This "symmetry rule" translates the configurational isomers of heme-site ligation into six observed switchpoints of quaternary transition. Cooperativity arises from both "concerted" quaternary switching and "sequential" modulation of binding within each quaternary form, T and R. Binding affinity is regulated through a hierarchical code of tertiary-quaternary coupling that includes the classical allosteric models as limiting cases.

Allosteric Regulation↗
PubMed1976

An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.

Bacterial Proteins↗
PubMed1985

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors.

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.

Base Sequence↗
PubMed1995

Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts.

Animals↗
PubMed1996

Transcript mapping and processing of mitochondrial RNA in the chlorophyte alga Prototheca wickerhamii.

The detailed transcript map of the circular 55328 bp mitochondrial (mt) genome from the colourless chlorophycean alga Prototheca wickerhamii has been determined. On each half of this genome the genes are encoded only on one DNA strand, forming transcriptional units comprising variable numbers of genes. With the exception of four genes coding for ribosomal proteins, transcripts of the three rRNA genes and all protein-coding genes have been detected by both northern analysis and primer extension experiments. Polycistronic transcripts of protein coding and tRNA genes were verified by northern analyses, primer extension and RNAse mapping experiments. The 5' and 3' ends of different RNA species are often located in close proximity to putative stem-loop structures and some 5' termini of mRNAs coincide with the 3' end of tRNAs located immediately upstream. Transcript mapping in a putative promoter region revealed two different possible transcription initiation sites; no significant sequence homology to putative mt promoters from higher plants could be found. In addition, two out of three group I introns residing in the cox1 gene were found to be self-splicing in vitro under reaction conditions developed for related mt introns from a filamentous fungus. Mitochondrial gene expression of P. wickerhamii and of filamentous fungi has several features in common, such as intron splicing and the processing of longer polycistronic transcripts. The similarities in RNA maturation between higher-plant and P. wickerhamii mitochondria are less pronounced, since plants rarely use tRNAs as processing signals for their relatively short mitochondrial co-transcripts.

Base Sequence↗