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Results for “insertion, inversion”

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PubMed2001

Molecular analysis of the mouse S100A9 gene and evidence that the myeloid specific transcription factor C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils.

The genomic locus of the mouse S100A9 (MRP14) gene, a myeloid expressed gene belonging to the S100 family, is split in three exons and two introns. Insertions of B1 like and LINE elements as well as several sequence repeat structures are scattered over the gene suggesting that this region of the S100 gene cluster has been the subject of a high mutational activity in mouse evolution. The insertions may represent molecular footprints of a recently postulated inversion event, which resulted in a rearrangement of the S100 gene cluster in mouse compared to man. Deletion analysis of the promoter reveals, that a 1200 bp fragment is able to direct a cell type-specific expression of a reporter gene in granulocytic 32D cells. Unexpectedly, the myeloid-specific transcription factor C/EBPepsilon is not needed for the transcriptional upregulation of the S100A9 and S100A8 genes in neutrophils. The data described here provide further insights into the evolution of the S100 gene cluster and into the myeloid-specific regulation of the murine S100A9 gene expression.

Animals↗

PubMed1980

[Origin of components of the transcallosal potential in the motor cortex].

The potentials of positive--negative configuration alone inverted on insertion of the leading electrode into the cortex. The inversion of responses in the point symmetrical to stimulation site occurred at the level of the II--II layers; in the point of maximal activity--at the V layer. The negative-positive transcallosal potentials as well as extracallosal responses did not invert. Transcallosal potentials of different configuration seem to be due to interaction of several flows of excitation within the cortex of recipient hemisphere. Inhibition processes seem to participate in forming the focus of transcallosal excitation.

Animals↗

PubMed1999

Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum.

Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so-called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.

Base Sequence↗

PubMed2006

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a.

BACKGROUND: Shigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401) and compared it with S. flexneri 2a (Sf301). RESULTS: The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI). Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS). There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes. CONCLUSION: Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.

Chromosome Aberrations↗

PubMed2005

A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease.

Diverse long interspersed element-1 (LINE-1 or L1)-dependent mutational mechanisms have been extensively studied with respect to L1 and Alu elements engineered for retrotransposition in cultured cells and/or in genome-wide analyses. To what extent the in vitro studies can be held to accurately reflect in vivo events in the human genome, however, remains to be clarified. We have attempted to address this question by means of a systematic analysis of recent L1-mediated retrotranspositional events that have caused human genetic disease, with a view to providing a more complete picture of how L1-mediated retrotransposition impacts upon the architecture of the human genome. A total of 48 such mutations were identified, including those described as L1-mediated retrotransposons, as well as insertions reported to contain a poly(A) tail: 26 were L1 trans-driven Alu insertions, 15 were direct L1 insertions, four were L1 trans-driven SVA insertions, and three were associated with simple poly(A) insertions. The systematic study of these lesions, when combined with previous in vitro and genome-wide analyses, has strengthened several important conclusions regarding L1-mediated retrotransposition in humans: (a) approximately 25% of L1 insertions are associated with the 3' transduction of adjacent genomic sequences, (b) approximately 25% of the new L1 inserts are full-length, (c) poly(A) tail length correlates inversely with the age of the element, and (d) the length of target site duplication in vivo is rarely longer than 20 bp. Our analysis also suggests that some 10% of L1-mediated retrotranspositional events are associated with significant genomic deletions in humans. Finally, the identification of independent retrotranspositional events that have integrated at the same genomic locations provides new insight into the L1-mediated insertional process in humans.

Base Sequence↗

PubMed1989

Cloning and characterization of the scarlet gene of Drosophila melanogaster.

DNA from the scarlet (st) region of Drosophila melanogaster has been cloned by chromosome walking, using the breakpoints of a new X-ray-induced third chromosome inversion (In(3LR)st-a27) which breaks in the scarlet (73A3.4) and rosy (87D13-14) regions. Two spontaneous mutants of st(st1 and stsp) contain insertions of non-st DNA located within 3.0 kb of the site of the inversion breakpoint used to isolate the gene, and a second scarlet inversion breaks within 6.5 kb of this site. However no changes detectable by Southern blotting were found in 5 X-ray-induced st mutants with cytologically normal third chromosomes. A 2.3-kb transcript arising from the st gene region (as defined by mutant analysis and DNA transformation) has been detected. This transcript is present throughout development at low levels, with a peak level during the early to mid-pupal stage. The size and amount of this transcript is altered in st1, and its amount is drastically reduced in stsp. Flies carrying the white1 mutation show normal levels of expression of the st transcript, suggesting that the w+ gene does not regulate transcription of the st+ gene. Nucleotide homology between sequences from the st transcription unit and a fragment carrying coding information from the white gene has been detected. This suggests that the st and w proteins are related; they appear to belong to a family of membrane-spanning, ATP-binding proteins involved in the transport of pigment precursors into cells.

Amino Acid Sequence↗

PubMed1996

Molecular characterization of hobo-mediated inversions in Drosophila melanogaster.

The structure of chromosomal inversions mediated by hobo transposable elements in the Uc-1 X chromosome was investigated using cytogenetic and molecular methods. Uc-1 contains a phenotypically silent hobo element inserted in an intron of the Notch locus. Cytological screening identified six independent Notch mutations resulting from chromosomal inversions with one breakpoint at cytological position 3C7, the location of Notch. In situ hybridization to salivary gland polytene chromosomes determined that both ends of each inversion contained hobo and Notch sequences. Southern blot analyses showed that both breakpoints in each inversion had hobo-Notch junction fragments indistinguishable in structure from those present in the Uc-1 X chromosome prior to the rearrangements. Polymerase chain reaction amplification of the 12 hobo-Notch junction fragments in the six inversions, followed by DNA sequence analysis, determined that each was identical to one of the two hobo-Notch junctions present in Uc-1. These results are consistent with a model in which hobo-mediated inversions result from homologous pairing and recombination between a pair of hobo elements in reverse orientation.

Animals↗

PubMed2004

A case of infantile acute lymphoblastic leukemia presenting with rearrangement of MLL at 11q23 and apparent insertion or translocation at 10p12.

We report the case of an 11-month-old patient with a clinical diagnosis of infantile acute lymphoblastic leukemia and an MLL (11q23) rearrangement in 69% of nuclei, revealed with interphase fluorescence in situ hybridization (FISH). Routine chromosome analysis of the bone marrow showed a very subtle rearrangement involving the short arm of chromosome 10 and the long arm of chromosome 11 in the abnormal cells. To clarify the nature of this rearrangement, we hybridized the MLL break-apart probe to previously G-banded slides. The rearrangement was interpreted as a small inversion within the band 11q23, separating the 5' MLL from the 3' MLL region. This segment on the long arm of chromosome 11 containing the rearranged MLL locus was either inserted in or translocated to the short arm of chromosome 10 at approximately band 10p12. The inversion affecting MLL may have followed insertion or preceded it. Molecular characterization of this rearrangement was not possible, due to limited sample material. There have been previous reports of rearrangements of MLL with the MLLT10 (alias AF10) gene locus at 10p12, including an interstitial inverted insertion of 11q13q23 in one case and insertion of 11q14q23 at 10p12 in another. These both resulted in a large derivative chromosome 10 and transcription of an MLL/MLLT10 fusion product. To our knowledge, the novel and cryptic rearrangement detected in our patient has not been described previously. A follow-up study of the patient's bone marrow at the end of induction therapy showed no morphologic evidence of residual leukemia and both FISH and chromosome analyses were normal.

Chromosomes, Human, Pair 10↗

PubMed1996

Possible role of splice acceptor site in expression of unspliced gag-containing message of Moloney murine leukemia virus.

Moloney murine leukemia virus (MLV) having the gag coding region alone, G3.6, produced a low level of mRNA (1/10 of the wild-type level). Ligation of 441 nucleotides (nt) containing a splice acceptor (SA) site to the downstream portion of the remaining gag region restored the level of the unspliced message, simultaneously activating a cryptic splice donor (SD) site in the middle of the p30 coding region (between nt 1596 and 1597). Ligation of the 441 nt in the same site in the inverted orientation also increased the level of the unspliced message, activating the same SD site (between nt 1596 and 1597) and a new SA site just in front of the inserted 441 nt (between nt 4770 and 4771). Deletion or inversion of the 441-nt SA sequence from the wild-type MLV or from int in-frame deletion or int frameshift mutant MLVs of nearly full size resulted in the loss of spliced mRNA and concomitantly in a severe reduction of the unspliced mRNA, particularly at 37 degrees C. Deletion of the 5' SD site did not result in the reduction of the unspliced-mRNA level. When the gag region in G3.6 was replaced with a Neo(r) coding region, the level of expression was high. The data taken together suggest that the presence of an SA signal is necessary for high-level expression of unspliced mRNA encoding Gag or Gag-Pol.

3T3 Cells↗

PubMed2001

Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes.

The ability to generate tagged mutants of Rhodococcus spp. will facilitate a deeper understanding of this medically and commercially important genus. The absence of efficient transposon systems in these organisms has here been overcome by the use of Tn5-based DNA-protein transposition complexes which can transpose at high efficiency. To achieve this, electroporation efficiencies and antibiotic selection were optimized. A Rhodococcus rhodochrous CW25 Tn5 insertion library of 1500 mutants was created. Southern blotting of 23 representative mutants demonstrated random insertion. A number of auxotrophic mutants were isolated and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Transposition of Tn5 was confirmed by the presence of 9 bp direct repeats of Rhodococcus DNA flanking the transposon insertion site. To further test this system, a Tn5 insertion library was constructed in a wild-type soil isolate of Rhodococcus spp. This is the first viable transposon knockout system reported for Rhodococcus.

Base Sequence↗

PubMed2000

Genes involved in the determination of the rate of inversions at short inverted repeats.

BACKGROUND: Not all of the enzymatic pathways involved in genetic rearrangements have been elucidated. While some rearrangements occur by recombination at areas of high homology, others are mediated by short, often interrupted homologies. We have previously constructed an Escherichia coli strain that allows us to examine inversions at microhomologies, and have shown that inversions can occur at short inverted repeats in a recB,C-dependent fashion. RESULTS: Here, we report on the use of this strain to define genetic loci involved in limiting rearrangements on an F' plasmid carrying the lac genes. Employing mini-Tn10 derivatives to generate insertions near or into genes of interest, we detected three loci (rmuA,B,C) that, when mutated, increase inversions. We have mapped, cloned and sequenced these mutator loci. In one case, inactivation of the sbcC gene leads to an increase in rearrangements, and in another, insertions near the recE gene lead to an even larger increase. The third gene involved in limiting inversions, rmuC, has been mapped at 86 min on the E. coli chromosome and encodes a protein of unknown function with a limited homology to myosins, and some of the SMC (structural maintenance of chromosomes) proteins. CONCLUSIONS: This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.

Chromosome Inversion↗

PubMed1993

Reducing attrition bias with an instrumental variable in a regression model: results from a panel of rheumatoid arthritis patients.

This study proposes an econometric technique to reduce attrition bias in panel data. In the simplest case, one estimates two regressions. The first is a probit regression based on sociodemographic and clinical characteristics measured at baseline. The probit regression estimates the probability that subjects stay or leave over the duration of the study. We insert the predicted probabilities from the probit regression into an inverse Mills ratio (IMR) or hazard rate to form an instrumental variable. We use this instrumental variable subsequently as an additional covariate in a second regression model that attempts to explain fluctuations in the dependent variable. The second regression, which is linear, includes only subjects who remained in the study. In alternative models, instrumental variables are created using predicted values from least squares and logit regressions estimating the probability that subjects stay or leave. The use of the instrumental variables reduces the effects of attrition bias in the linear regression model. We applied the technique to a panel of patients with rheumatoid arthritis (RA) enrolled in 1981 and followed through 1990. We attempted to predict values for a measure of functional disability recorded in 1990 with use of covariates measured in 1981. The dependent variable was an index of disability in 1990 and the independent variables (covariates) included the disability index from 1981, the years of duration of RA, gender, marital status, education, and age in 1981. The correction technique suggested that ignoring attrition bias would underestimate the strength of associations between being female and the subsequent disability index, and overestimate the strength of associations between being married spouse present, age, and the initial disability index on the one hand and the subsequent disability index on the other.

Age Factors↗

PubMed1997

Structural and functional analyses of DNA bending induced by Sp1 family transcription factors.

DNA bending induced by eukaryotic transcription factors may play a direct role in the activation of transcription by bringing together factors bound at non-adjacent sites or facilitate binding of factors involved in the formation of an initiation complex. The ubiquitously expressed zinc-finger transcription factor Sp1 is involved in the regulation of a variety of viral and cellular genes. During the past few years proteins homologous to Sp1 have been described constituting a family of Sp1 transcription factors. We have used gel electrophoretic methods to analyse the extent, location and direction of the DNA bend induced by four different Sp1 family proteins upon binding to a consensus GC box. We found that the Sp1 family proteins induce an asymmetric bend in DNA directed towards the major groove, with a bend centre displaced towards the 3' end of the GC box. The zinc-finger domain was alone responsible for introducing this distortion. The magnitude of the induced bend varied between the different proteins. Construction of a hybrid protein and mutation of the 3' end of the GC box indicated that zinc finger 1 is important both for the magnitude of the bend angle, location of the bend centre and the binding affinity. Transactivation studies of a Sp1-dependent promoter revealed that a 5 bp insertion between the TATA box and the GC box, or inversion of the GC box significantly reduced the promoter activity, indicating that protein-induced bending could be important for promoter activity. However, no stimulatory effect could be observed in cotransfections with the DNA binding domain of Sp1 in Drosophila SL-2 cells, suggesting that the bending activity alone is not sufficient for transactivation.

Amino Acid Sequence↗

PubMed2002

Chiral mutagenesis of insulin's hidden receptor-binding surface: structure of an allo-isoleucine(A2) analogue.

The hydrophobic core of vertebrate insulins contains an invariant isoleucine residue at position A2. Lack of variation may reflect this side-chain's dual contribution to structure and function: Ile(A2) is proposed both to stabilize the A1-A8 alpha-helix and to contribute to a "hidden" functional surface exposed on receptor binding. Substitution of Ile(A2) by alanine results in segmental unfolding of the A1-A8 alpha-helix, lower thermodynamic stability and impaired receptor binding. Such a spectrum of perturbations, although of biophysical interest, confounds interpretation of structure-activity relationships. To investigate the specific contribution of Ile(A2) to insulin's functional surface, we have employed non-standard mutagenesis: inversion of side-chain chirality in engineered monomer allo-Ile(A2)-DKP-insulin. Although the analogue retains native structure and stability, its affinity for the insulin receptor is impaired by 50-fold. Thus, whereas insulin's core readily accommodates allo-isoleucine at A2, its activity is exquisitely sensitive to chiral inversion. We propose that the Ile(A2) side-chain inserts within a chiral pocket of the receptor as part of insulin's hidden functional surface.

Binding Sites↗

PubMed1997

DNA replication promotes high-frequency homologous recombination during Autographa californica multiple nuclear polyhedrosis virus infection.

The relative ease with which foreign genes can be incorporated into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) indicates that a highly efficient recombinational process exists within infected cells. However, it is unclear whether this is due to marker transfer mediated by host cell enzymes or recombination events promoted by AcMNPV itself. To address the latter possibility, a pair of inverted repeat IS50 elements derived from the bacterial transposon Tn5 was inserted into the polyhedrin gene locus of the AcMNPV genome. Inversion of Tn5 sequences arising from recombination between its IS50 repeats could be readily detected in this virus, indicating that AcMNPV DNA undergoes high-frequency recombination during infection. To further characterize this process, a transient recombination assay was developed and used to identify the cis- and trans-acting requirements for Tn5 inversion in AcMNPV. A transfected Tn5-containing plasmid was found to undergo the same sequence inversion events seen in the viral genome, but only if it also contained a putative AcMNPV origin of replication (homologous region 2) in cis and was replicated by AcMNPV gene products supplied in trans. Taken together, these results indicated that recombination events which occur in infected cells were strictly dependent upon AcMNPV-mediated DNA replication. Direct support for this hypothesis was provided by the observation that the minimal set of AcMNPV genes that was essential for plasmid DNA replication also promoted recombination events leading to Tn5 inversion in the absence of any other viral function. Finally, using a panel of deletion mutants of the IS50 elements in Tn5, sequence inversion was shown to be the result of homologous rather than site-specific recombination, since it occurred independently of a discrete sequence within the transposon. These results demonstrate that the AcMNPV DNA replication machinery exhibits a strong propensity to promote homologous recombination events during infection and is likely to play a role in the high frequency of marker transfer observed in this virus.

Animals↗

PubMed1983

Mode of insertion of miconazole, ketoconazole and deacylated ketoconazole in lipid layers. A conformational analysis.

The conformation of three imidazole derivatives, miconazole, ketoconazole and deacylated ketoconazole (R 39 519) inserted in a lipid layer was calculated using a procedure of conformational analysis. For each imidazole derivative all probable conformers were inserted into a dipalmitoyl phosphatidylcholine (DPPC) monolayer. Miconazole maintains its two dichlorophenyl groups in the hydrophobic phase whereas the imidazole moiety is orientated in the hydrophilic phase. Ketoconazole orientates its dichlorophenyl group in the hydrophobic phase whereas its acylated piperazine moiety is orientated towards the hydrophobic region. Deacylation inverses completely the orientation of the compound. The most probable conformer of R 39 519 is inserted in the lipid layer with its piperazine moiety orientated towards the aqueous phase. The inversion increases the area occupied per drug molecule from 30 A2 for ketoconazole to 90 A2 for R 39 519 equal to the mean area occupied per miconazole molecule and higher than that occupied per DPPC molecule (60 A2). Such a conformation should result in a destabilizing effect of miconazole and R 39 519; this was proved using differential scanning calorimetry.

Antifungal Agents↗

PubMed2006

Characterization of human Rab20 overexpressed in exocrine pancreatic carcinoma.

In a large-scale analysis of gene expression in pancreatic cancer, we isolated the homologue of the mouse Rab20. The mouse protein was previously identified during a search for novel Rab proteins, a family of small GTP-binding proteins involved in the regulation of intracellular vesicular transport. The Rab20 protein has no close relationship to any member of the Rab protein subfamily. In contrast to other members, it contains an insertion of 40 amino acids of unknown function and an inversion of 3 amino acids at the position corresponding to codon 61 in p21ras proteins. Using immunofluorescence and immunoelectron microscopy, we localized the Rab20 protein in the vicinity of the Golgi apparatus. Rab20 expression was detected by Western blot analysis in 11 of 11 pancreatic tumor cell lines and 7 of 8 primary pancreatic carcinomas. Absent or very faint expression was observed in normal pancreas cell extracts. Immunohistochemical analysis of Rab20 in tissues showed low or absent expression in normal pancreas and stronger expression in 15 of 18 exocrine pancreatic adenocarcinomas. Rab20 was also detected in preneoplastic pancreatic intraductal neoplasia lesions, suggesting that its up-regulation may be an early event in pancreatic carcinogenesis.

Adenocarcinoma↗

PubMedDate not supplied

Walking tree heuristics for comparative genomic alignments.

Genomic sequence data is available for an ever-increasing number of organisms, but the full meaning of this data remains an enigma. String alignment is one approach for deciphering the information contained in genetic strings. Sequences which are conserved across species will help identify genes and other important structures. Similarity between species can be scored by measuring how well their sequences align. The walking tree method is an approximate string alignment method that can handle insertions, deletions, substitutions, translocations, and more than one level of inversion. We will describe this method and recent improvements which allow fast alignment of megabase strings. We will show examples in which the method located or discovered genes. We show how the method can be used to construct phylogenetic trees. We also show that the method can be used to identify essential regions for protein function.

Animals↗