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A method for construction of E. coli strains with multiple DNA insertions in the chromosome.

A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage lambda, has been developed. Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these fragments into the chromosome of int-Tn+ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage lambda xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the lambda attL and lambda attR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.

Chromosomes, Bacterial↗

Isolation and characterization of a plasmid from Treponema denticola.

Agarose gel electrophoresis of whole genomic DNA of the oral spirochaete Treponema denticola has revealed a plasmid-like fraction. Purification and restriction enzyme analysis has confirmed the presence of a 2.6-kb circular plasmid, which has been mapped for restriction sites and cloned into the Escherichia coli plasmid pUC18. Southern blot analysis of genomic T. denticola DNA, using the plasmid as a probe, has shown that the plasmid is present only as an extra-chromosomal element. No plasmid-coded recombinant gene product from a PstI insert in pUC18 has been detected in host cells of E. coli by SDS-PAGE or immunoblotting with polyclonal immune rabbit serum to T. denticola. The discovery of this plasmid may provide a useful tool in the application of new molecular approaches in spirochaetal biology.

Blotting, Southern↗

Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca(2+)-binding site using a tryptophan mutant.

Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.

Amino Acid Sequence↗

Rapid and efficient site-specific mutagenesis without phenotypic selection.

Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

Base Sequence↗

Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.

Active Transport, Cell Nucleus↗

Stability of patch methylation and its impact in regions of transcriptional initiation and elongation.

CpG DNA methylation has previously been correlated with the suppression of transcription. The mechanism of this suppression is not understood, and many aspects of the temporal and positional relationships between the region of methylation and transcription have not yet been defined. Here, 12-kb stable replicating episomes that can be maintained in human somatic cells for weeks to months were used. Such a system allows more direct manipulation and is free from the positional effects attendant with the analysis of endogenous loci or integrated transgenes. By using these circular minichromosomes, patches of CpG methylation were created to include or exclude the regions of transcriptional initiation and elongation. I found that a 0.5-kb patch of methylation that covered the promoter suppressed expression only 2-fold and that a 1.9-kb patch of methylation that covered the coding portion of the gene (but not the promoter) suppressed expression about 10-fold. In contrast, methylation of the entire minichromosome except for the promoter or the coding portion suppressed transcription about 50- to 200-fold. I infer the following. Methylation of the 0.5-kb promoter fragment does not significantly affect transcription at the level of transcription factor binding or local chromatin structure. The dominant effect on transcription occurs when the length of methylated DNA is long, with little disproportionate effect of methylation of specific regions, such as that of initiation or elongation. I also found that the boundaries between these methylated and unmethylated regions remained stable for the many weeks that I monitored them.

Cell Line↗

First complete mitochondrial genome of Uzelothrips scabrosus (Thysanoptera: Uzelothripidae) provides insights into gene rearrangements and phylogenetic position within Terebrantia.

The family Uzelothripidae is represented by a single genus Uzelothrips and can be distinguished from others by the presence of whip-like antennae, a circular ventral sensorium on antennal segment III, a well-developed tentorium, and a membranous ovipositor. Here, we generated the first complete mitochondrial genome of Uzelothrips scabrosus (15,674 bp) using next-generation sequencing to explore the gene rearrangements and phylogenetic relationships. It consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and two putative control regions. The genome exhibits strong AT bias (71.35%) with negative AT and GC skew. Codon usage analyses indicate a strong bias towards A/U-ending codons and influenced by both natural selection and mutation pressure. All PCGs were under purifying selection, with cox1 being the most conserved and nad4L the most variable. The gene order of the family Uzelothripidae is highly rearranged compared to the ancestral insect gene order. Comparative analysis revealed that gene block B was the most widely conserved, whereas the remaining gene blocks exhibited family or lineage-specific conservation patterns, reflecting extensive mitochondrial gene rearrangements during the evolution of the Thysanoptera. Moreover, 228 synapomorphic and 68 autapomorphic gene boundaries were identified across thysanopteran mitogenomes. Phylogenies indicated that the family Uzelothripidae is in a sister relationship with Stenurothripidae, and the Uzelothripidae + Stenurothripidae clade is sister to Thripidae. This study provides the first mitogenomic insights into Uzelothripidae and highlights the need for broader taxon sampling and nuclear genomic data to resolve deep evolutionary relationships within Thysanoptera.

Comparative analysis↗

OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts.

We have developed a vector, OVEC ("oligonucleotide vector") to study DNA sequences involved in the regulation of transcription in mammalian cells. This vector is equally suitable for studying expression in vivo after transfection into cells, or for transcription studies in vitro with cell-free extracts. Putative cis-acting DNA segments from enhancers or promoters can be inserted at a position immediately upstream of the TATA box and coding sequence of the rabbit beta-globin gene. A regulatory DNA segment can be tested by itself or in conjunction with an enhancer located either in an adjacent upstream position, or downstream of the beta-globin gene. S1 nuclease mapping can be used to study transcription from circular and linear templates and run-off transcription in vitro is also feasible. Transcripts from a reference globin gene with a small deletion around the transcription initiation site can be measured with the same S1 nuclease probe and thus serve as an internal standard. We demonstrate the usefulness of OVEC by inserting either short oligonucleotides comprising a metal responsive enhancer element, or the SV40 enhancer, directly upstream of the TATA box. Both constructs yield high levels of correctly initiated transcripts in a transient expression assay in HeLa cells. In a HeLa cell nuclear extract the SV40 enhancer stimulates transcription 40-fold.

Animals↗

Movement characteristics and acrosomal status of rabbit spermatozoa recovered at the site and time of fertilization.

Rabbit spermatozoa were recovered from the oviductal ampullae 11 h postcoitus by an oil microflush technique. Their movement was evaluated in the ampullar fluid, or in ampullar fluid diluted with in vitro fertilization medium, in slide preparations which were approximately 25 micron or 100 micron deep. The movement of these sperm was compared with the movement of ejaculated sperm in diluted semen. Movement parameters measured from videotapes recorded by a high-speed camera were coded according to treatment and entered into a microcomputer for statistical analysis. A total of 157 spermatozoa were recovered from the oviducts of 16 does: 152 were motile and 126 were free-swimming. Nearly all of the free-swimming sperm swam in trajectories whose average paths were circular. The flagellar beat pattern of the circular swimmers was asymmetric and nearly planar, and the sperm did not roll. Spermatozoa observed in 25-micron slide preparations produced smaller flagellar bends than sperm swimming in 100-micron preparations and tended to swim in larger circles which were oriented in the plane of the slide. Spermatozoa observed within the cumulus matrix moved in a slow, erratic, sinuous manner, but resumed rapid circling upon leaving the matrix. It was concluded that the ampullar sperm were hyperactivated, retaining this physiological condition as they entered the cumulus. The movement qualitatively resembled that of hyperactivated guinea pig and hamster spermatozoa because these species effectively swim in circles. In contrast, 80% of the ejaculated spermatozoa swam in linear trajectories, resulting from relatively symmetrical, flagellar beat patterns. The percentage of rolling spermatozoa and the rolling frequencies were less in the 25-micron than the 100-micron slide preparations. Thus, the movement parameters of both ampullar and ejaculated spermatozoa were affected by the geometry of their observation chambers. This influence should be taken into account when observing sperm motility in vitro. It could also be important in vivo, where changes in sperm movement in response to epithelial surfaces might provide an advantage for reaching the cumulus mass. Ninety-eight percent of the motile ampullar sperm were observed to have acrosomes, including all spermatozoa found within the cumulus matrix.

Acrosome↗

Nucleotide sequence of a circular single-stranded DNA associated with coconut foliar decay virus.

A circular single-stranded (ss) covalently closed (ccc) DNA associated with coconut foliar decay virus (CFDV) was purified, amplified by the polymerase chain reaction, and subcloned and its sequence established by analysis of overlapping subgenomic cDNA clones. The complete CFDV sequence comprised 1291 nucleotides and contained open reading frames for six proteins of molecular weight larger than 5 kDa. One of these (ORF1, 33.4 kDa) codes for a leucine-rich protein with the nucleoside triphosphate-binding motif GXGKS and may possibly participate in virus replication. The putative viral protein encoded by ORF3 (6.4 kDa) is a positively charged arginine-rich protein with homology to the capsid protein of nuclear polyhedrosis virus, and may represent the CFDV coat protein. CFDV DNA can form a stable stem structure of 10 GC base pairs subtending a loop sequence which in one orientation closely resembles the motif TAATATTAC conserved in a similar structural arrangement within the geminivirus group. Otherwise no sequence homology to DNA-containing plant viruses of the gemini- or caulimovirus groups was found. CFDV therefore represents a new taxonomic group of plant viruses.

Amino Acid Sequence↗

Direction of transcription affects the replication mode of lambda in an in vitro system.

A set of artificial circular plasmids, named plasmoids, was constructed. They are about 1 kb in size and consist of a 178 bp lambda dv minimal DNA replication origin (ori) which has four direct repeats and the A + T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac). They carry no other functional genes or genetic sites. The constructions involved various combinations of relative orientations of these components. These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al. 1981), and can be used for studies of interactions between transcription and replication. In these plasmids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites. The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand. The most active replication took place when the major RNA synthesis went through the lambda origin region in the direction which occurs naturally in the lambda genome. Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription. When RNA synthesis through the lambda origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak.

Bacteriophage lambda↗

Genetic engineering, isolation and characterization of a truncated Escherichia coli elongation factor Tu comprising domains 2 and 3.

A deletion mutant of a plasmid born Escherichia coli tufA gene, which codes for a truncated elongation factor Tu comprising domains 2 and 3, has been constructed by genetic engineering. This gene was overexpressed in E. coli, and a polypeptide representing the truncated elongation factor Tu was isolated, purified to near homogeneity, crystallized and characterized physico-chemically as well as biochemically. Circular dichroism spectroscopy and limited tryptic digestion demonstrate that the isolated domain pair 2 and 3 behaves like an independent folding unit which adopts a similar secondary and most likely, tertiary, structure to that present in the intact elongation factor Tu. However, the isolated domain pair 2 and 3 does not interact with aminoacyl-tRNA or the antibiotic kirromycin, two ligands which were shown previously by cross-linking experiments to be in contact with amino acid residues located in domains 1 and 2, and domain 3, respectively. The results suggest that the isolated domain pair 2 and 3 by itself forms too few contacts with these ligands to form a stable complex. Furthermore, the data suggest that domain 1 in intact EF-Tu, in a subtle but nevertheless decisive manner, alters the conformation of the other two domains in such a way that all three domains cooperatively create a high affinity binding site for aminoacyl-tRNA and the antibiotic kirromycin.

Circular Dichroism↗

Resolution of a polyomavirus-mouse hybrid replicon: release of genomic viral DNA.

RmI is a circular chimera containing 1.03 copies of polyomavirus DNA and 1,628 base pairs of mouse DNA, joined through direct and inverted repeat sequences. It is excised from the chromosome of a transformed cell via a site-specific recombination event that is dependent on the activation of the viral gene coding for large T antigen. RmI is shown here to be highly infectious for normal mouse cells. This infectivity reflects the ability of RmI to effectively yield unit-length viral DNA via intramolecular recombination. The effectiveness with which infectious viral DNA is produced from RmI is consistent with the idea that the underlying recombination event is site specific, rather than homologous or illegitimate.

Animals↗

Neurons in the supplementary eye field of rhesus monkeys code visual targets and saccadic eye movements in an oculocentric coordinate system.

1. We investigated whether neurons in the supplementary eye field (SEF) of macaque monkeys code saccadic eye movements in oculocentric coordinates (relative to the current direction of fixation) or in craniocentric coordinates (relative to the head). Craniocentric coding in SEF had been previously suggested by the convergent appearance of electrically elicited saccades originating at different orbital positions. 2. We primarily studied SEF neurons that started responding before the beginning of saccades because such presaccadic activity is likely related to saccade generation and metrics. Using a memory-saccade task, we classified the presaccadic activity of each neuron as either purely visual related, purely movement related, or both visual and movement related. 3. We then mapped the response fields (receptive fields and movement fields) of SEF neurons from different orbital positions. When mapped relative to a central fixation point, the strongest responses for a given SEF neuron invariably occurred for a particular polar direction with fairly symmetrical declines for departures from that direction. When tested using other fixation point locations, their strongest responses almost always continued to occur for stimuli having the same polar direction relative to each fixation point tested, and thus they appeared to code both stimulus direction and saccade direction in an oculocentric coordinate system. 4. The effect of eye position on SEF presaccadic activity was quantified in two ways by computing, for each neuron, 1) an "intersection distance," the eccentricity of the point where extensions of the neuron's optimal polar directions measured at two eccentric orbital positions converged, and 2) an "orbital perturbation index" such that an index of 0 corresponded to no change in the neuron's optimal polar direction across different orbital positions (i.e., perfectly oculocentric response fields) and an index of 1 corresponded to optimal polar directions that converged to the same craniocentric goal regardless of initial eye position (i.e., perfectly craniocentric response fields). For neurons with both visual and movement responses, these measures were calculated separately for each type of activity using tasks that temporally separated the visual cue presentation and the saccade to it. 5. Almost all of the intersection distances were well beyond the oculomotor range (+/- 50 degrees) of the monkey (38/39 for movement activity and 62/66 for visual activity). The median intersection distance for visual activity was very large (274 degrees), and the median for movement activity was slightly divergent (beyond infinity). Thus SEF neurons rarely showed a conspicuous convergence of response field direction. 6. Likewise, the mean orbital perturbation indexes were very small (-0.04 +/- 0.21, mean +/- SD, for movement activity and 0.09 +/- 0.15 for visual activity), also indicating that SEF neurons code stimuli and saccades in an oculocentric manner. 7. For neurons with both visual and movement activities, the orbital perturbation indexes of the two activities were not significantly correlated (r = 0.16), even though their characteristic directions (optimal polar direction estimated from the center of the screen) were almost the same (circular correlation, r+ = 0.97). The lack of a significant correlation between the visual and movement activity orbital perturbation indexes is consistent with the hypothesis that most of the variation in this index represents statistically independent errors of measurement. Conversely, the strong covariation of visual and movement activity characteristic directions indicates that directional preference is a fundamental functional property of SEF presaccadic activity.(ABSTRACT TRUNCATED)

Animals↗

Basic helix-loop-helix proteins E2A and HEB induce immature T-cell receptor rearrangements in nonlymphoid cells.

T-cell receptor (TCR) gene rearrangements are mediated via V(D)J recombination, which is strictly regulated during lymphoid differentiation, most probably through the action of specific transcription factors. Investigated was whether cotransfection of RAG1 and RAG2 genes in combination with lymphoid transcription factors can induce TCR gene rearrangements in nonlymphoid human cells. Transfection experiments showed that basic helix-loop-helix transcription factors E2A and HEB induce rearrangements in the TCRD locus (Ddelta2-Ddelta3 and Vdelta2-Ddelta3) and TCRG locus (psi Vgamma7-Jgamma2.3 and Vgamma8-Jgamma2.3). Analysis of these rearrangements and their circular excision products revealed some peculiar characteristics. The Vdelta2-Ddelta3 rearrangements were formed by direct coupling without intermediate Ddelta2 gene segment usage, and most Ddelta2-Ddelta3 recombinations occurred via direct coupling of the respective upstream and downstream recombination signal sequences (RSSs) with deletion of the Ddelta2 and Ddelta3 coding sequences. Subsequently, the E2A/HEB-induced TCR gene recombination patterns were compared with those in early thymocytes and acute lymphoblastic leukemias of T- and B-lineage origin, and it was found that the TCR rearrangements in the transfectants were early (immature) and not necessarily T-lineage specific. Apparently, some parts of the TCRD (Vdelta2-Ddelta region) and TCRG genes are accessible for recombination not only in T cells, but also in early B-cells and even in nonlymphoid cells if the appropriate transcription factors are present. The transfection system described here appeared to be useful for studying the accessibility of immunoglobulin and TCR genes for V(D)J recombination, but might also be applied to study the induction of RSS-mediated chromosome aberrations.

Animals↗

Plasmid functions involved in the stable propagation of the pKD1 circular plasmid in Kluyveromyces lactis.

Plasmid factors involved in the stable propagation of pKD1-derived vectors in Kluyveromyces lactis transformants have been identified. Three genes (A, B and C) have been found to be present in pKD1: the interruption of the B and C genes led to high plasmid instability. Stability could be restored in trans when host cells contained pKD1 as the resident plasmid (pKD1+ strains). The A gene, which codes for a site-specific recombinase, did not affect plasmid partitioning. Vectors bearing only the pKD1 replication origin (or a chromosomal ARS), and no other pKD1 sequence, were very unstable both in the presence and absence of the resident plasmid in host cells. These vectors could be stabilized in pKD1+ strains, but not in pKD1 degree strains, by the insertion of a 200 bp-long pKD1 sequence. This sequence, called the cis-acting stability locus (CSL), together with the products of the B and C genes, ensured plasmid partitioning at cell division. Possible hairpin structures and direct repeats were regularly spaced within the CSL. This region, and the corresponding cis-acting stabilizing elements of other yeast plasmids, did not have sequence homology but shared some structural regularities.

Base Sequence↗

Conformational changes upon binding of a receptor loop to lipid structures: possible role in signal transduction.

The mas oncogene codes for a seven transmembrane helix protein. The amino acid sequence 253-266, from the third extracellular loop and beginning of helix 7, was synthesized either blocked or carrying an amino acid spin label at the N-terminus. Peptide binding to bilayers and micelles was monitored by ESR, fluorescence and circular dichroism. Binding induced tighter lipid packing, and caused an increase of peptide secondary structure. While binding to bilayers occurred only when peptide and phospholipid bore opposite charges, in micelles the interaction took place irrespective of charge. The results suggest that changes in lipid packing could modulate conformational changes in receptor loops related to the triggering of signal transduction.

Amino Acid Sequence↗

Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases.

The cloning in Escherichia coli of the 3' moieties of the lytA and cpl-1 genes is described, coding for the C-terminal regions of the lytic amidase of Streptococcus pneumoniae and the phage Cp-1 lysozyme, respectively. The truncated genes were overexpressed in E. coli and the purified polypeptides showed a great affinity for choline, although they were devoid of cell wall-degrading activity. Biochemical and circular dichroism analyses indicated that these are the domains responsible for the specific recognition of the choline-containing pneumococcal cell walls by the lytic enzymes. The data presented here suggested that these choline-binding domains can function independently of their catalytic domains.

Amidohydrolases↗