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At least 577 records · Page 32Linked to original sources
PubMed1995

Inversion of the IDS gene resulting from recombination with IDS-related sequences is a common cause of the Hunter syndrome.

We have recently described the identification of a second IDS locus (IDS-2) located within 90 kb telomeric of the IDS gene (Bondeson et al. submitted). Here, we show that this region is involved in a recombination event with the IDS gene in about 13% of patients with the Hunter syndrome. Analysis of the resulting rearrangement at the molecular level showed that these patients have suffered a recombination event that results in a disruption of the IDS gene in intron 7 with an inversion of the intervening DNA. Interestingly, all of the six cases with a similar type of rearrangement showed recombination between intron 7 of the IDS gene and sequences close to exon 3 at the IDS-2 locus implying that these regions are hot spots for recombination. Analysis by nucleotide sequencing showed that the inversion is caused by recombination between homologous sequences present in the IDS gene and the IDS-2 locus. No detectable deletions or insertions were observed as a result of the recombination event. The results in this study have practical implications for diagnosis of the Hunter syndrome.

Base Sequence↗
PubMed1996

Phylogenetic affinities of the grasses to other monocots as revealed by molecular analysis of chloroplast DNA.

The distribution of structural alterations of the chloroplast genome found in grass chloroplast (cp) DNA in comparison with that of tobacco was systematically surveyed in the cpDNAs of monocots. Southern hybridization and/or PCR analyses for the detection of (1) three inversions in the large single-copy region, (2) loss of an intron in the rpoC1 gene, (3) an extra-sequence insertion in the rpoC2 gene, (4) the deletion of ORF2280, (5) rearrangements of the accD (ORF512) gene, and (6) non-reciprocal translocation of the rpl23 gene, were carried out on cpDNAs isolated from 58 species, 22 families, and 11 orders, which covered almost all families of monocots. These structural alterations of cpDNA mostly occurred at the family level. However, only part of the Restionaceae possessed the inversion that characterizes the lineage of grass differentiation. The order of mutational events made it possible to reconstruct grass phylogeny in monocots. Since no variations in structural alterations of the cpDNA were found among the Poaceae, grass plants were inferred to have originated from an ancestor harboring these structural alterations of the chloroplast genome. These phylogenetic relationships were supported by the sequence data of rbcL.

Base Sequence↗
PubMed1994

Analysis of a complex plasmid insertion in a phototaxis-deficient transformant of Dictyostelium discoideum selected on a Micrococcus luteus lawn.

A novel method for clonal selection of G418-resistant Dictyostelium discoideum transformants on lawns of Micrococcus luteus was developed. The procedure was used to isolate transformants deficient in phototaxis after nontargeted insertion of shuttle vector DNA. Southern blot analysis as well as restriction, T-tracking, and sequencing analysis of plasmids rescued from the genomic DNA of one of the putative phototaxis gene disruptants showed that it contained a complex multicopy insertion of the vector. While insertions of such plasmid vectors might typically be in tandem multicopy format, they can be much more complex, containing, as in this case, inverse as well as tandemly duplicated copies and various deletions.

Animals↗
PubMed1993

PilS and PilR, a two-component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa.

Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12,700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial-specific bacteriophage PO4. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage-resistant mutants was identified in which the transposon had inserted near sequences that cross-hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN-dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild-type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72-75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA-D. PilS and PilR clearly belong to the family of two-component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.

Amino Acid Sequence↗
PubMed1991

Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS.

Escherichia coli pilG mutants are thought to have a dramatically higher DNA inversion rate as measured by the site-specific DNA inversion of the type 1 pili pilA promoter. DNA sequence of the pilG gene confirmed its identity to the gene encoding the bacterial histonelike protein H-NS. Unlike other histonelike protein complexes that enhance site-specific DNA recombination, the H-NS protein inhibited this process. This inhibition was indicated by the increased inversion rate of the pilA promoter region effected by two different mutant pilG alleles. One of these alleles, pilG1, conferred a mutant phenotype only at low temperature attributable to a T-to-G transversion in the -35 sequence of the pilG promoter. The other allele, pilG2-tetR, was an insertion mutation in the pilG coding region that conferred the mutant phenotype independent of temperature. We measured an approximately 100-fold-increased pilA promoter inversion rate in the mutant by exploiting the temperature-dependent expression of pilG1 and using a novel rapid-population-sampling method. Contrary to one current view on how the H-NS protein might act to increase DNA inversion rate, we found no evidence to support the hypothesis that DNA supercoiling affected pilA promoter inversion.

Alleles↗
PubMed2004

Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster.

Flytrap is a web-enabled relational database of transposable element insertions in Drosophila melanogaster. A green fluorescent protein (GFP) artificial exon carried by a transposable P-element is mobilized and inserted into a host gene intron creating a GFP fusion protein. The sequence of the tagged gene is determined by sequencing inverse-PCR products derived from genomic DNA. Flytrap contains two principle data types: micrographs of protein localization and a cellular component ontology, based on rules derived from the Gene Ontology consortium (http://www.geneontology.org), describing protein localization. Flytrap also has links to gene information contained in Flybase (http:// flybase.bio.indiana.edu). The system is designed to accept submissions of micrographs and descriptions from any type of tissue (e.g. wing imaginal disk, ovary) and at any stage of development. Insertion lines can be searched using a number of queries, including Berkeley Drosophila Genome Project (BDGP) numbers and protein localization. In addition, Flytrap provides online order forms linked to each insertion line so that users may request any line generated from this project. Flytrap may be accessed from the homepage at http://flytrap.med. yale.edu.

Animals↗
PubMed1992

Analysis of X-ray-induced HPRT mutations in CHO cells: insertion and deletions.

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) for the presence or absence of exons revealed that the causes of the mutant phenotype were total gene deletion (26/41), partial gene deletion (4/41), and an insertion (1/41). No alterations of exon number or sizes were apparent in 10 of the mutants. Southern blot analysis confirmed the deletion data and revealed an additional class of mutants that had a gene disruption but retained all hprt exons (2/41). Therefore, at least 80% of the ionizing radiation-induced mutations were due to mechanisms involving DNA breakage and rejoining. The distribution of deletion sizes suggests that the two DNA breaks required for a deletion are not independent events. A possible mechanism is presented. In addition, the DNA sequence of the insertion mutation was determined. The insertion (229 bp) is coupled with a deletion (31 bp). An imperfect inverted repeat with flanking hprt DNA was identified and may be involved in the insertion event.

Animals↗
PubMed1996

Segregation of a paternal insertional translocation results in partial 4q monosomy or 4q trisomy in two siblings.

A genetics evaluation was requested for a 6-week-old infant with multiple congenital malformations including mild craniofacial anomalies, truncal hypotonia, hypospadias, and a ventriculoseptal defect. Blood obtained for chromosome analysis revealed an abnormal chromosome 4. Paternal chromosome analysis showed a 46,XY, inv ins (3;4)(p21.32;q25q21.2), inv(4)(p15.3q21.2) karyotype. Therefore, the proband's chromosome 4 was the unbalanced product of this insertional translocation from the father resulting in partial monosomy 4q. Additionally, the derivative 4 had a pericentric inversion which was also seen in the father's chromosome 4. During genetic counseling, the proband's 2-year-old brother was evaluated. He was not felt to be abnormal in appearance, but was described as having impulsive behavior. Chromosome analysis on this child revealed 46,XY,der(3)inv ins(3;4)(p21.32;q25q21.2)pat. This karyotype results in partial trisomy 4q. FISH using two-color "painting" probes for chromosomes 3 and 4 confirmed the G-banded interpretation in this family. The segregation seen in this family resulted in both reciprocal products being observed in the two children, with partial 4q monosomy showing multiple congenital anomalies, and partial 4q trisomy showing very few phenotypic abnormalities.

Child, Preschool↗
PubMed1990

Serial subculture and relative transport of human endothelial cells in serum-free, defined conditions.

Endothelial cells bind, process, and transport bioactive molecules and thus provide an interactive interface between the plasma and adjacent tissues. Various hormones and factors induce endothelial cells to synthesize and secrete interactive factors. However, study of endothelial cell synthesis, processing, and transport of these bioactive molecules has been impeded because of the serum requirement for cell growth. Many of these bioactive molecules are derived from or are modified by serum components. We have developed a short-term culture system that supports sequential subculturing of endothelial cells in a serum-free culture medium on a defined extracellular matrix. The cells have a doubling rate of 33 h and the total cell number can be expanded more than 800-fold. Expression of specific markers; factor VIII related antigen-von Willebrand factor, Wiebel-Palade bodies, a cobblestone appearance of confluent cell monolayers, and angiotensin-converting enzyme activity confirm the normal morphologic integrity and biochemical function of these cultures. Using this defined serum-free medium, we have grown human endothelial cell monolayers on porous polycarbonate membrane inserts, thereby creating an upper and a lower chamber that models the vascular architecture and demonstrates an inverse relationship between transport and molecular weight. By eliminating serum components, this model system should facilitate the study of endothelial cell binding, metabolism, and transport of bioactive molecules and may contribute to a better understanding of the blood-tissue interface.

Angiotensin II↗
PubMed2002

Biodiversity (Communications arising): maize transgene results in Mexico are artefacts.

Quist and Chapela's conclusion that the transgenes they claim to have detected in native maize in Oaxaca, Mexico, are predominantly reassorted and inserted into a "diversity of genomic contexts" seems to be based on an artefact arising from the inverse polymerase chain reaction (i-PCR) they used to amplify sequences flanking 35S transgenes from cauliflower mosaic virus (CaMV).

Artifacts↗
PubMed2003

Site-specific recombination by the DDE family member mobile element IS30 transposase.

DNA rearrangements carried out by site-specific recombinases and transposases (Tpases) show striking similarities despite the wide spectrum of the catalytic mechanisms involved in the reactions. Here, we show that the bacterial insertion sequence (IS)30 element can act similarly to site-specific systems. We have developed an inversion system using IS30 Tpase and a viable lambda phage, where the integration/excision system is replaced with IS30. Both models have been proved to operate analogously to their natural counterpart, confirming that a DDE family Tpase is able to fulfill the functions of site-specific recombinases. This work demonstrates that distinction between transposition and site-specific recombination becomes blurred, because both functions can be fulfilled by the same enzyme, and both types of rearrangements can be achieved by the same catalytic mechanisms.

Amino Acid Motifs↗
PubMed1975

Site specific recA--independent recombination between bacterial plasmids: involvement of palindromes at the recombinational loci.

A recA-independent recombinational event is described which results in insertion of an entire plasmid genome at a unique site of another plasmid, and coincident excision of a precisely defined DNA segment originally present at the point of the insertion. The resulting recombinant molecules subsequently can undergo site-specific translocation of their component segments or inversion of their original DNA sequence orientation. The events observed entail nonreciprocal exchange of genetic material, and involve a discrete nucleotide sequences that is duplicated in rotationally symmetrical reverse orientation on plasmid DNA (i.e., inverted repeat; palindrome).

Base Sequence↗
PubMed2004

Elements in the N-terminal signaling sequence that determine cytosolic topology of short-chain dehydrogenases/reductases. Studies with retinol dehydrogenase type 1 and cis-retinol/androgen dehydrogenase type 1.

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11beta-hydroxysteroid dehydrogenase (11beta-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11beta-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.

Alcohol Oxidoreductases↗
PubMed1995

Molecular organization and germinal instability of R-stippled maize.

The spotted seed allele R-stippled (R-st) is comprised of the following genetic components: strong seed color (Sc), inhibitor-of-R (I-R) and near-colorless seed (Nc). I-R is a mobile element that represses (Sc) expression irregularly. Germinal I-R losses produce progeny with fully colored seed. Southern blot analysis revealed four r-hybridizing segments in R-st and three, two or one in two sets of unequal crossover deletion products. By comparison to published reports of r gene structure, we maintain that each segment contains at least one r gene. The proximal r gene, Sc, confers strong seed color; the three distal r genes together produce near-colorless seed. R-st's seed spotting phenotype is correlated with the presence of a 3.3-kb insert in Sc identified as I-R. The level of the near-colorless phenotype is inversely correlated with the number of r genes present, suggesting involvement of a multiple copy silencing mechanism in their regulation. Phenotypic changes in R-st occurred primarily by unequal exchange between r genes. The locations of exchange positions showed a strong polarity, nearly all occurring in the 3' portions of the identified r genes.

Alleles↗
PubMed2003

Svi3: A provirus common integration site in c-myc in SL/Kh pre-B lymphomas.

Spontaneous pre-B lymphomas in SL/Kh mice occur by somatic acquisition of a provirus genome of endogenous murine leukemia virus (MuLV). Inverse PCR amplification and sequence analyses of a provirus and its host flanking fragment revealed a proviral insertion into c-myc in 3 out of 60 SL/Kh pre-B lymphomas, named Svi3 lymphomas (SL/Kh virus integration site-3). Southern blot analysis revealed that two lymphomas had clonal integration in c-myc exon 1 and the other, in the promoter region. In 2 out of 3 Svi3 lymphomas, a fusion transcript of provirus 3' long terminal repeat and c-myc and a normal full-length c-myc transcript were obtained, but in one Svi3 lymphoma, only the normal transcript was obtained. All three Svi3 lymphomas had increased c-myc expression, producing normal 67-kDa c-Myc protein. Svi3 lymphomas had more mature phenotypes in the steps of early B-cell differentiation than Svi1 lymphomas, in which c-myc expression was indirectly up-regulated by provirus integration into Stat5a.

3' Untranslated Regions↗
PubMed2000

The mechanical waveform of the basilar membrane. III. Intensity effects.

Mechanical responses in the basal turn of the guinea-pig cochlea were measured with broad-band noise stimuli and expressed as input-output cross-correlation functions. The experiments were performed over the full range of stimulus intensities in order to try to understand the influence of cochlear nonlinearity on frequency selectivity, tuning, signal compression and the impulse response. The results are interpreted within the framework of a nonlinear, locally active, three-dimensional model of the cochlea. The data have been subjected to inverse analysis in order to recover the basilar-membrane (BM) impedance, a parameter function that, when inserted into the (linearized version of that) model, produces a model response that is similar to the measured response. This paper reports details about intensity effects for noise stimulation, in particular, the way the BM impedance varies with stimulus intensity. In terms of the underlying cochlear model, the decrease of the "activity component" in the BM impedance with increasing stimulus level is attributed to saturation of transduction in the outer hair cells. In the present paper this property is brought into a quantitative form. According to the theory [the EQ-NL theorem, de Boer, Audit. Neurosci. 3, 377-388 (1997)], the BM impedance is composed of two components, both intrinsically independent of stimulus level. One is the passive impedance Zpass and the other one is the "extra" impedance Zextra. The latter impedance is to be multiplied by a real factor gamma (0 < or = gamma < or = 1) that depends on stimulus level. This concept about the composition of the BM impedance is termed the "two-component theory of the BM impedance." In this work both impedances are entirely derived from experimental data. The dependence of the factor gamma on stimulus level can be derived by using a unified form of the outer-hair-cell transducer function. From an individual experiment, the two functions Zpass and Zextra are determined, and an approximation (Zpass + gamma Zextra) to the BM impedance constructed. Next, the model response (the "resynthesized" response) corresponding to this "artificial" impedance is computed. The same procedure is executed for several stimulus-level values. For all levels, the results show a close correspondence with the original experimental data; this includes correct prediction of the compression of response amplitudes, the reduction of frequency selectivity, the shift in peak frequency and, most importantly, the preservation of timing in the impulse response. All these findings illustrate the predictive power of the underlying model.

Animals↗
PubMed1992

Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis.

A novel, chromosomally located conjugative transposon in Lactococcus lactis, Tn5276, was identified and characterized. It encodes the production of and immunity to nisin, a lanthionine-containing peptide with antimicrobial activity, and the capacity to utilize sucrose via a phosphotransferase system. Conjugal transfer of Tn5276 was demonstrated from L. lactis NIZO R5 to different L. lactis strains and a recombination-deficient mutant. The integration of Tn5276 into the plasmid-free strain MG1614 was analyzed by using probes based on the gene for the nisin precursor (nisA) and the gene for sucrose-6-phosphate hydrolase (sacA). The transposon inserted at various locations in the MG1614 chromosome and showed a preference for orientation-specific insertion into a single target site (designated site 1). By using restriction mapping in combination with field inversion gel electrophoresis and DNA cloning of various parts of the element including its left and right ends, a physical map of the 70-kb Tn5276 was constructed, and the nisA and sacA genes were located. The nucleotide sequences of Tn5276 junctions in donor strain NIZO R5 and in site 1 of an MG1614-derived transconjugant were determined and compared with that of site 1 in recipient strain MG1614. The results show that the A + T-rich ends of Tn5276 are flanked by a direct hexanucleotide repeat in both the donor and the transconjugant but that the element does not contain a clear inverted repeat.

Base Sequence↗