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Results for “insertion, inversion”

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PubMed1996

Identification and isolation of the FEEBLY gene from tomato by transposon tagging.

The Ac/Ds transposon system from maize was used for insertional mutagenesis in tomato. Marker genes were employed for the selection of plants carrying a total of 471 unique Ds elements. Three mutants were obtained with Ds insertions closely linked to recessive mutations: feebly (fb), yellow jim (yj) and dopey (dp). The fb seedlings produced high anthocyanin levels, developed into small fragile plants, and were insensitive to the herbicide phosphinothricin. The yj plants had yellow leaves as a result of reduced levels of chlorophyll. The dp mutants completely or partially lacked inflorescences. The fb and yj loci were genetically linked to the Ds donor site on chromosome 3. Reactivation of the Ds element in the fb mutants by crosses with an Ac-containing line resulted in restoration of the wild-type phenotypes. Plant DNA fragments flanking both sides of the Ds element in the fb mutant were isolated by the inverse polymerase chain reaction. Molecular analysis showed that phenotypic reversions of fb were correlated with excisions of Ds. DNA sequence analysis of Fb reversion alleles showed the characteristic Ds footprints. Northern and cDNA sequence analysis indicated that transcription of the FEEBLY (FB) gene was impeded by the insertion of Ds in an intron. Comparison of the predicted amino acid sequence of the FB protein with other database sequences indicated that FB is a novel gene.

Aminobutyrates↗

PubMed1994

Foot inversion-eversion and knee kinematics during walking.

The purpose of this study was to monitor selected aspects of the three-dimensional kinematics of the knee during walking with regular shoes and with modified shoes that induced either pronation or supination of the foot. Steinmann traction pins were inserted into the right tibia and femur of five adult men who had apparently normal lower extremities. Target clusters mounted onto the pins were filmed by four cine cameras operating at 100 frames/sec. Two trials per subject were analyzed for each of the three experimental conditions: regular running shoes, running shoes with a 10 degree valgus wedge, and running shoes with a 10 degree varus wedge. The different types of footwear induced only minor kinematic changes at the knee during the stance phase of walking. The angular patterns of the tibiofemoral joint were modified by less than 1 degree, whereas the translatory patterns were altered by 2 mm. Immediately following foot-strike, the valgus-wedge shoes caused the tibia to rotate internally 4 degrees more than the varus-wedge shoes, but at the tibiofemoral joint no consistent differences in the pattern of internal-external rotation between normal and modified footwear were measureable. These findings suggest that, in the healthy lower extremity, increased internal and external tibial rotation is resolved at the hip joint, with changes at the tibiofemoral joint that barely are detectable with the techniques used in this study.

Adult↗

PubMed1997

Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS.

The H-NS protein is a major component of the Escherichia coli nucleoid. Mutations in hns, the gene encoding H-NS, have pleiotropic effects on the cell altering both the expression of a variety of unlinked genes and the inversion rate of the DNA element containing the fimA promoter. We investigated the interaction between H-NS and fimB, the gene encoding the bidirectional recombinase that catalyzes fimA promoter flipping. In beta-galactosidase assays, we found that fimB expression increased approximately fivefold in an hns2-tetR insertion mutant. In gel mobility shift assays with purified H-NS, we have also shown that H-NS bound directly and cooperatively to the fimB promoter region with greater affinity than for any other known H-NS-regulated gene. Furthermore, this high-affinity interaction resulted in a promoter-specific inhibition of fimB transcription. The addition of purified H-NS to an in vitro transcription system yielded a fivefold or greater reduction in fimB-specific mRNA production. However, the marked increase in cellular FimB levels in the absence of H-NS was not the primary cause of the mutant rapid inversion phenotype. These results are discussed in regard to both H-NS as a transcriptional repressor of fimB expression and its role in regulating type 1 pilus promoter inversion.

Bacterial Proteins↗

PubMed1989

Addition of molecular methods to mutation studies with Drosophila melanogaster.

For 80 years, Drosophila melanogaster has been used as a major tool in analyzing Mendelian genetics. By using chromosome inversions that suppress crossing over, geneticists have developed a large number of stocks for mutation analysis. These stocks permit numerous tests for specific locus mutations, lethals at multiple loci on any chromosome, chromosome exchanges, insertions, and deletions. The entire genome can be manipulated for a degree of genetic control not found in other germ-line systems. Recombinant DNA techniques now permit analysis of mutations to the nucleotide level. By combining classical genetic analysis with recombinant DNA techniques, it is possible to analyze mutations that range from chromosome aberrations and multilocus deficiencies to single nucleotide transitions.

Animals↗

PubMed1995

An intron facilitates activation of the calspermin gene by the testis-specific transcription factor CREM tau.

Calspermin is a high affinity Ca2+/calmodulin binding protein that is found only in postmeiotic male germ cells. Our previous studies have shown that the calspermin transcript is produced by utilization of a testis-specific promoter located within an intron of the calmodulin kinase IV gene. This promoter contains two cAMP response element-like motifs that bind the testis-specific transcription factor CREM tau. This interaction is required for transcriptional activation. Here we describe a novel regulatory element, the 111-base pair first intron of the calspermin gene, which is also required for enhancement of transcription by CREM tau via the cAMP response element motifs. Deletion or inversion of this intron results in loss of CREM tau-mediated stimulation of transcription. However, CREM tau stimulates calspermin promoter activity when the intron is moved upstream of the promoter but only when inserted in the proper orientation. Footprint, linker scanning, and deletion analyses were used to identify regulatory elements in the intron. We suggest that the intron functions as an orientation-dependent but position-independent regulatory element to activate the calspermin promoter by facilitating the stimulatory effect of CREM tau on transcription.

3T3 Cells↗

PubMed1993

Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions.

Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.

Base Sequence↗

PubMed2003

Comparative study of the FAIR technique of perfusion quantification with the hydrogen clearance method.

Arterial spin labeling magnetic resonance methods, including flow-sensitive alternating inversion recovery (FAIR), are becoming increasingly common for the noninvasive quantification of cerebral blood flow (CBF). This report compares the FAIR method with hydrogen clearance. The latter is an established, invasive technique for CBF measurement in animals. Paired readings of CBF were obtained in gerbils to maximize the degree of spatial and temporal correspondence between methods. Flow-sensitive alternating inversion recovery (50 averages, 6.7-minute measurement time) and hydrogen clearance measurements were made concurrently. Cerebral blood flow values measured by both techniques displayed an initial decrease because of the injurious effects of electrode insertion and subsequent recovery. Mixed model regression analysis, structural equations modeling, and a simple concordance correlation coefficient analysis were performed. No evidence of a marked systematic bias in the FAIR measurements was found; mixed model regression analysis yielded relative bias estimates of 0.4 (confidence interval: 3.0, 3.9) mL. 100 g-1. min-1 and -3.7 (-12.1, 4.7) mL. 100 g-1. min-1 at 20 and 100 mL. 100 g-1. min-1, respectively. The principal limitation of the FAIR technique was the magnitude of the random measurement error (imprecision), which had a standard deviation on the order of 10 mL. 100 g-1. min-1.

Animals↗

PubMed2005

Radiofrequency catheter ablation of common atrial flutter in a patient with anomalous inferior vena cava and azygos continuation.

We describe a case of isthmus-dependant atrial flutter ablation with a superior approach due to an anomalous inferior vena cava (IVC), azygos, and emiazygos continuation. A 56-year-old man was referred to our institution for the treatment of a common atrial flutter. Two years prior, the patient had an attempted electrophysiological study not performed due to the inability to advance the guidewire through IVC. A computed tomography of the abdominal venous system revealed the absence of the subrenal IVC, azygos, and emiazygos continuation. Following double puncture of the left subclavian vein, two catheters were inserted into the coronary sinus and into the right atrium. Programmed atrial stimulation was performed and a sustained isthmus-dependant right atrial tachycardia was obtained. After 11 applications of radiofrequency energy with an inversion technique, sinus rhythm was restored and a repeat atrial programmed stimulation failed to induce any arrhythmia.

Atrial Flutter↗

PubMed1999

Structural unbalanced chromosome rearrangements resolved by comparative genomic hybridization.

The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis.

Abortion, Spontaneous↗

PubMed2005

[Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation].

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.

Animals↗

PubMed2006

Characterisation of 96 mutations in 128 unrelated severe haemophilia A patients from France. Description of 62 novel mutations.

Direct sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII<1%) in 128 unrelated haemophiliacs A, negative for intron 22 and intron 1 inversions. A mutation was found in 122/128 patients (95%). Ninety-six distinct mutations were identified in this cohort, 62 of these are novel. They consisted of deletions (7 large and 24 small deletions), insertions (n = 9), associations of insertion/deletion (n = 2), association of deletion/substitution (n = 1), and single nucleotide substitutions (53 point mutations consisting of 31 missense, 20 nonsense, and 2 splicing mutations). Twenty-two patients had developed inhibitors, and among this subgroup 3 large deletions, 6 frameshift, 9 nonsense and 4 missense mutations were detected. For 6 patients, among which one developed an anti-FVIII inhibitor, no mutations were detected in the coding and splicing regions of factor VIII gene. Different approaches of molecular modelling were performed in addition to familial linkage analysis to determine the pathophysiological responsibility of these novel missense mutations.

Blood Coagulation Tests↗

PubMed1990

Nucleotide sequence of the maize chloroplast rpo B/C1/C2 operon: comparison between the derived protein primary structures from various organisms with respect to functional domains.

The genes (rpo B/C1/C2) coding for the beta, beta', beta" subunits of maize (Zea mays) chloroplast RNA polymerase have been located on the plastome and their nucleotide sequences established. The operon is part of a large inversion with respect to the tobacco and spinach chloroplast genomes and is flanked by the genes trnC and rps2. Notable features of the nucleotide sequence are the loss of an intron in rpoC1 and an insertion of approximately 450 bp in rpoC2 compared to the dicotyledons tobacco, spinach and liverwort. The derived amino acid sequence of this additional monocotyledon specific sequence is characterized by acidic heptameric repeat units containing stretches of glutamic acid, tyrosines and leucines with regular spacing. Other structural motifs, such as a nucleotide binding domain in the beta subunit and a zinc finger in the beta' subunit, are compared at the amino acid level throughout the RNA polymerase subunits with the enzymes from other organisms in order to identify functionally important conserved regions.

Amino Acid Sequence↗

PubMed1999

Estimating loop-helix interfaces in a polytopic membrane protein by deletion analysis.

Insertions of amino acids into transmembrane helices of polytopic membrane proteins disrupt helix-helix interactions with loss of function, while insertions into loops have little effect on transmembrane helices and therefore little effect on activity [Braun, P., Persson, B., Kaback, H. R., and von Heijne, G. (1997) J. Biol. Chem. 272, 29566-29571]. Here the inverse approach, amino acid deletion, is utilized systematically to approximate loop-helix boundaries in the lactose permease of Escherichia coli. Starting with deletion mutants in the periplasmic loop between helices VII and VIII (loop VII/VIII), which has been defined by immunological analysis and nitroxide-scanning electron paramagnetic resonance spectroscopy, it is shown that mutants with single or multiple deletions in the central portion of the loop retain significant transport activity, while deletion of amino acid residues near the loop-helix boundaries or within the flanking helices leads to complete inactivation. Results consistent with hydropathy analysis are obtained with loops VI/VII, VIII/IX, and IX/X and the flanking helices. In contrast, deletion analysis of loops III/IV, IV/V, and V/VI and the flanking helices indicates that this region of the permease differs from hydropathy predictions. More specifically, evidence is presented supporting the contention that Glu126 and Arg144 which are charge paired and critical for substrate binding are within helices IV and V, respectively.

Amino Acid Sequence↗

PubMed1994

Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping.

The technique of promoter trapping has been exploited to identify markers of embryogenesis in Arabidopsis thaliana. A population of transgenic A. thaliana was generated containing the promoter trap vector pdeltagusBin19, following Agrobacterium tumefaciens-mediated transformation. This vector contains at the T-DNA left border, a promoterless gusA gene, which is activated following integration downstream of a native gene promoter that directs transgene transcription. Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimetric assay. Histochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to contain single T-DNA inserts were analysed in detail. Each line showed a distinct pattern of GUS fusion activity. Fusion transcripts were identified, demonstrating that transcription was initiated in the genomic DNA flanking the T-DNA left border. Inverse PCR was used to clone the T-DNA flanking sequences, and for one line a corresponding cDNA was identified, demonstrating that the tagged sequences are transcribed. The markers represent the earliest embryonic genes known to be expressed in plants.

Arabidopsis↗

PubMed1990

Derivation of clones from the choroideremia locus by preparative field inversion gel electrophoresis.

By making use of preparative field inversion gel electrophoresis, we have constructed a lambda ZAP library that is highly enriched for sequences from the choroideremia locus. In vivo excision of pBluescript SK(-) constructs from lambda ZAP obviates the subcloning of DNA inserts and allows for rapid processing of several hundred recombinants. From a 625 kb Sfil fragment we isolated 7 clones that were physically mapped using microdeletions associated with the disease. One of these clones is located within, or just telomeric to, the choroideremia gene and detects two restriction fragment length polymorphisms (RFLPs). Another clone detects a RFLP which maps centromeric to the disease locus. Together these probes should improve the reliability of linkage analysis in choroideremia families and should pave the way for the isolation of the choroideremia gene.

Animals↗

PubMed1989

Dispersed repeats and structural reorganization in subclover chloroplast DNA.

The plastid genome from subclover, Trifolium subterraneum, is unusual in a variety of respects, compared with other land-plant chloroplast DNAs. Gene mapping of subclover chloroplast DNA reveals major structural reorganization of the genome. Ten clusters of genes are rearranged in both order and orientation. Eight large inversions are sufficient to explain this reorganization; however, the actual evolutionary changes may have been more complex. For example, a fine-scale analysis of a set of ribosomal protein genes reveals the occurrence of insertions, deletions, and transpositions. Associated with this unusually unstable genome are two structural features potentially involved in the rearrangements. A dispersed family of repeats, with each element about 1 kb in length, is present in at least six copies. A survey of a wide taxonomic range of species indicates that these elements are unique to the chloroplast DNAs of subclover and two closely related species. Several of the repeated elements are associated with genomic rearrangements, and one repeat is inserted within a normally highly conserved series of genes. This set of dispersed repeats may be the first family of transposable elements found in any organelle genome. In addition, the subclover genome is much larger than those in other closely related legumes, even when one takes into account the presence of the repeated elements. Some of the extra DNA has no sequence similarity to other chloroplast genomes and may represent insertion of DNA from another genome. These unusual features are not found in the structurally stable chloroplast genomes of other vascular plants and may, therefore, be implicated in the rapid and major reorganization of the chloroplast DNA in subclover.

Biological Evolution↗

PubMed1995

Complete sequence of a mariner transposable element from the predatory mite Metaseiulus occidentalis isolated by an inverse PCR approach.

Degenerate primers designed and synthesized based on two conserved regions of the mariner transposase open reading frame were used to amplify a 454 bp DNA fragment from M. occidentalis. Two inverse primers were then synthesized and used to amplify flanking genomic DNA fragments from M. occidentalis by a ligation-mediated inverse PCR. The complete mariner element (Moc1) was 1284 bp long, including the imperfect 28 bp inverted terminal repeat sequences, and shared 59% similarity to an active 1286 bp long D. mauritiana mariner element (Mos1). Insertions, deletions and substitutions were observed in the Moc1 sequence at several positions. No intact open reading frame was detected and the Moc1 element is considered inactive. Stringent Southern blot hybridizations revealed at least twelve copies of mariner sequences similar to Moc1 in the colonies tested.

Animals↗

PubMed1996

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.

Animals↗