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PubMed1986

A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli.

The vir region of Ti plasmids is responsible for the transfer of the T region from Agrobacteria to plant cells; previous experiments suggested that formation of independent T region DNA circles is one step in this process. To study this step in Escherichia coli, we developed a binary vector system. One plasmid (= substrate) contains correctly oriented right and left borders from octopine plasmid pTiAch5. A gene with a counterselectable function (galK) was cloned between these borders. The galK gene is under control of the tac promoter-operator and the lac repressor with the laci gene also in the selection cassette. This construction allows determination of substrate plasmid mutants which have lost the selectable galK function. The second component of the system is one of a set of compatible plasmids harbouring various cloned parts from the vir region of nopaline plasmid pTiC58. A 3.6-kbp segment of the vir region turned out to be necessary and sufficient for production of substrate plasmid mutants which represented the equivalent of the T region containing a complete left border. From this vir region fragment four discrete proteins were expressed in minicells. The coding regions were mapped to a part conserved in nopaline and octopine plasmids; in the latter it appears to correspond to virC/D.

Cloning, Molecular↗

PubMed1987

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↗

PubMed1990

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↗

PubMed2005

Protein fragment complementation in M.HhaI DNA methyltransferase.

The 5mC DNA methyltransferase M.HhaI can be split into two individually inactive N- and C-terminal fragments that together can form an active enzyme in vivo capable of efficiently methylating DNA. This active fragment pair was identified by creating libraries of M.HhaI gene fragment pairs and then selecting for the pairs that code for an active 5mC methyltransferase. The site of bisection for successful protein fragment complementation in M.HhaI was in the variable region near the target recognition domain between motif VIII and TRD. This same region is the location of bifurcation in the naturally split 5mC methyltransferase M.AquI, the location for circular permutation in M.BssHII, and the location for previously engineered split versions of M.BspRI.

Amino Acid Sequence↗

PubMed1987

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↗

PubMed1996

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↗

PubMed2001

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↗

PubMed1991

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↗

PubMed1995

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↗

PubMed1990

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↗

PubMed2004

Novel plastid gene minicircles in the dinoflagellate Amphidinium operculatum.

Seven new minicircles, forming part of the fragmented plastid genome of the dinoflagellate Amphidinium operculatum, have been identified by PCR. Three minicircles are full-length, one of 2.6 kb encoding the 23S rRNA gene, one of 2.4 kb containing the psaB gene, and a third of 2.5 kb containing the psbD, psbE and psbI genes. This is the first report of a three-gene minicircle. All three genes are conventional in length, with the same codon bias found in other minicircle genes. The psbI gene sequence is very divergent. One empty minicircle, of 1.7 kb, and three 'microcircles', between 400 and 600 bp, have also been identified by PCR. They appear to be formed from full-length minicircles by homologous recombination and internal deletion. All three microcircles contain the core region common to minicircles, but are missing a coding region, providing further evidence that the core region is necessary for minicircle replication and maintenance.

Amino Acid Sequence↗

PubMed2001

Film dosimetric verification of the Voxel Monte Carlo (VMC) algorithm with electron beam dose distributions.

Monte Carlo (MC) methods have the potential to predict radiation-therapy doses more accurately than any conventional technique, but normal MC simulations are very time consuming. Therefore, a fast MC code (Voxel Monte Carlo; VMC) was developed especially for radiation therapy purposes and experiments with the comparable precision were performed to demonstrate its accuracy. In the present study the dose distributions measured with film dosimetry in a cylindrical phantom were compared with calculations derived by VMC. The phantom consisted of 18 circular shaped PMMA slabs with a diameter of 20 cm and a thickness of approx. 1 cm. The films were placed between the slabs, and the whole phantom was irradiated with electron beams of different energies (6 MeV, 10 MeV, 18 MeV). The measured optical density distributions were then converted into dose distributions using characteristic curves of the film. Taking into account experimental uncertainties and statistical calculation fluctuations, agreement was found between measurements and VMC simulations with a maximal deviation of 3 mm on isodose curves for 18 MeV.

Algorithms↗

PubMed2004

The genome of phiAsp2, an actinoplanes infecting phage.

The first genome of a virus infecting a representative of the eubacterial genus Actinoplanes is presented. Phage phiAsp2 has a circularly permutated chromosome that consists of 58,638 bp; its G/C-bias of 70.39% resembles the hosts G + C-content (71-73% within the genus). A total of 76 open reading frames (orfs) were identified, the majority of which (63) displaying equal transcriptional orientations. Functional gene clustering is obvious as orfs coding for head and tail proteins are located close to the center in the first half of the genome and putative DNA-modifying enzymes are encoded by centrally located genes; DNA repair and recombination functions are situated in the remaining part of the genome, adjacent to a small gene cluster, the predicted proteins of which are involved in DNA packaging. Close to the left terminus there are two small regions (approximately 4.5 kb each, separated by 2.8 kb) which are homologous to the recently sequenced mycobacteriophage rosebush, however, the unique overall structure of the phiAsp2-genome does not bear resemblance to any other known viral genome. The nucleotide sequence was deposited in GenBank with the accession no. AY576796.

Acarbose↗

PubMed1998

Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension.

A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.

Arabidopsis↗

PubMed2003

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana.

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis: Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

Amino Acid Sequence↗

PubMed2025

The Complete Chloroplast Genome and the Phylogenetic Analysis of Panicum bisulcatum (Thumb.) (Poaceae).

The chloroplast (cp) genome of Panicum bisulcatum (Thumb.), a significant agricultural weed, was sequenced and characterized to elucidate its genomic architecture, evolutionary dynamics, and phylogenetic relationships. The complete cp genome was assembled as a circular DNA molecule of 138,489 bp, exhibiting a typical quadripartite structure comprising a large single-copy (LSC, 82,260 bp), a small single-copy (SSC, 12,569 bp), and a pair of inverted repeats (IR, 21,830 bp each) regions. It encodes 135 genes, including 89 protein-coding genes, 49 tRNAs, and 8 rRNAs. Functional annotation revealed that most genes are involved in photosynthesis and genetic system. A total of 51 simple sequence repeats (SSRs) and 62 long repeats (LRs) were identified, providing potential molecular markers. Comparative analysis of IR boundaries highlighted both conserved features and species-specific expansion/contraction events among Panicum species. Phylogenomic analysis robustly placed P. bisulcatum within the genus Panicum, showing a closest relationship with P. incomtum and confirming the monophyly of the genus. Furthermore, single nucleotide polymorphism (SNP) analysis with its closest relative, P. incomtum, revealed 4659 SNPs, with a dominance of synonymous substitutions, indicating the action of purifying selection. This study provides the first comprehensive cp genomic resource for P. bisulcatum, which will facilitate future studies in species identification, phylogenetic reconstruction, population genetics, and the development of sustainable management strategies for this weed.

Phylogeny↗

PubMed2002

Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy.

IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage lambda gt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly alpha-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.

Allergens↗

PubMed1998

Analysis of the genome of Mycobacterium tuberculosis H37Rv.

The powerful combination of genomics and bioinformatics is providing a wealth of information about Mycobacterium tuberculosis, the aetiological agent of human tuberculosis, that will facilitate the conception and development of new therapies. The starting point for genome sequencing was the integrated map of the 4.4 Mb circular chromosome of the widely used, virulent reference strain, M. tuberculosis H37Rv. Cosmids and bacterial artificial chromosomes were selected from ordered libraries and subjected to systematic shotgun sequence analysis. This approach simplified sequence assembly as the genome is rich in repetitive DNA. In common with most bacteria, > 90% of the potential coding capacity is used, and probable or tentative functions could be attributed to > 70% of the genes. The potential biological roles of two of the principal driving forces in genome dynamics, insertion sequence elements and polymorphic multigene families are discussed.

DNA Transposable Elements↗