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Results for “insertion, inversion”

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PubMed2003

Optimal induction of T-cell responses against hepatitis C virus E2 by antigen engineering in DNA immunization.

Although DNA immunization is a safe and efficient method for inducing cellular immune responses, it generates relatively weak and slow immune responses. Here, we investigated the effect of hepatitis C virus (HCV) antigen modifications on the induction of T-cell responses in DNA immunization. It is likely that the strength of T-cell responses has an inverse relationship with the length of the insert DNA. Interestingly, a mixture of several plasmids carrying each gene induced a higher level of T-cell responses than a single plasmid expressing a long polyprotein. Moreover, the presence of a transmembrane domain in HCV E2 resulted in stronger T-cell responses against E2 protein than its absence. Taken together, our results indicate that the tailored modifications of DNA-encoded antigens are capable of optimizing the induction of T-cell responses which is required for eliminating the cells chronically infected with highly variable viruses such as HCV and human immunodeficiency virus.

Animals↗

PubMed2002

Ahi-1, a novel gene encoding a modular protein with WD40-repeat and SH3 domains, is targeted by the Ahi-1 and Mis-2 provirus integrations.

The Ahi-1 locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-myb proto-oncogene. Since no significant alteration of c-myb expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1 locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3' end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1 gene. The Ahi-1 cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1 gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1 gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1 is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1 may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abl and c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abl defective retroviruses or a c-myc transgene or in tumors exhibiting deletion of Nf1.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor.

[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P functions as a low-potency antagonist but a high-potency full inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide, the C-terminal carboxyamidated pentapeptide wFwLX was identified as the core structure, which itself displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket [i.e., between transmembrane (TM)-III, TM-VI and TM-VII]. In contrast, the inverse agonist peptides bind in a pocket that extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI/VII to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, TM-IV and TM-V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deeply across the main ligand-binding pocket and sterically blocking the movement of TM-VI and TM-VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high-potency, selective inverse agonism that was dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also nonpeptide inverse agonists for the ghrelin receptor.

Amino Acid Sequence↗

PubMed2005

The genome of Salmonella enterica serovar gallinarum: distinct insertions/deletions and rare rearrangements.

Salmonella enterica serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:--:--) as S. enterica serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of S. enterica serovar Typhimurium LT2, which we used as a reference Salmonella genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing srfJ, std, and stj and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.

Bacterial Proteins↗

PubMed1991

In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.

Amino Acid Sequence↗

PubMed1986

Enhanced rate of conversion or recombination of markers within a region of unique sequence in the herpes simplex virus genome.

Insertion mutants of herpes simplex virus type 1, containing a second copy of the sequences of BamHI fragment L (map coordinates 0.706 to 0.744) inserted in inverted orientation into the thymidine kinase gene (at map coordinate 0.315), have been further characterized. We reported previously that, as a result of intramolecular or intermolecular recombination between copies of the BamHI-L sequence at the normal locus and inserted locus, a high proportion of progeny genomes exhibited either inversions of the unique sequence flanked by these inverted repeats or other rearrangements. Now we report that a genetic marker (syn-1 or syn-1+) originally present only in the inserted copy of BamHI fragment L appears in progeny at both the normal and inserted loci, and vice versa, at high frequency. Because these phenomena have not been observed with other insertion mutants containing duplications of other sequences from unique regions of the genome, we conclude that BamHI fragment L contains an element that enhances the rate of homologous recombination in adjacent sequences, resulting in genome rearrangements and gene conversion-like events.

Base Sequence↗

PubMed1983

The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli.

The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site. This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.

Base Sequence↗

PubMed2002

The association between HLA-A alleles and young Alu dimorphisms near the HLA-J, -H, and -F genes in workshop cell lines and Japanese and Australian populations.

At least two polymorphic Alu insertions have been previously identified and characterized within the class I region of the major histocompatibility complex (MHC). We have identified another two new polymorphic Alu insertions, AluyHJ and AluyHF, located near HLA-J and HLA-F, respectively, within the a block of the MHC. Here we report on (1). the haplotypic relationships between the Alu dimorphisms and the HLA-A locus within a panel of 51 IHW homozygous cell lines representing at least 36 HLA class I haplotypes, (2). the Alu genotype, allele, and haplotype frequencies present in the Australian Caucasians and Japanese populations, and (3). the frequency of association between the different Alu dimorphisms and the HLA-A alleles in 109 Australian Caucasians and 99 Japanese. PCR was used to detect the presence or absence of insertion for AluyHJ, AluyHG, and AluyHF within the DNA samples prepared from the cell lines and the two population groups that had been previously typed for HLA-A. In the homozygous cell lines, all three Alu insertions were found in only one HLA class I haplotype (HLA-A1, -B57, -Cw6), no Alu insertions were detected in six HLA class I haplotypes and one or more of the Alu insertions were found in 29 HLA class I haplotypes. At least one of the Alu insertions was found in about 86% of the Japanese and Australian individuals, with the AluyHJ generally related inversely to AluyHG and/or AluyHF. The gene frequency of the AluyHJ and AluyHF insertions was significantly different (p <0.05) between Japanese and Australians, whereas there was no difference (P > 0.05) between the frequencies of AluyHG in the two populations. The Alu haplotype frequencies were also significantly different between the Japanese and the Australians. In the cell lines and the population groups, the AluyHJ insertion was most frequently found associated with HLA-A1 or A24, AluyHG with HLA-A2, and AluyHF with HLA-A2, -A10, or -A26. This study suggests that the three polymorphic Alu elements have been inserted into the a block of the MHC in different progenitor groups and therefore will be useful lineage and linkage markers in human population studies and for elucidating the evolution of HLA class I haplotypes.

Alleles↗

PubMed2004

Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h.

Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.

DNA Primers↗

PubMed2001

Twin priming: a proposed mechanism for the creation of inversions in L1 retrotransposition.

L1 retrotransposons are pervasive in the human genome. Approximately 25% of recent L1 insertions in the genome are inverted and truncated at the 5' end of the element, but the mechanism of L1 inversion has been a complete mystery. We analyzed recent L1 inversions from the genomic database and discovered several findings that suggested a mechanism for the creation of L1 inversions, which we call twin priming. Twin priming is a consequence of target primed reverse transcription (TPRT), a coupled reverse transcription/integration reaction that L1 elements are thought to use during their retrotransposition. In TPRT, the L1 endonuclease cleaves DNA at its target site to produce a double-strand break with two single-strand overhangs. During twin priming, one of the overhangs anneals to the poly(A) tail of the L1 RNA, and the other overhang anneals internally on the RNA. The overhangs then serve as primers for reverse transcription. The data further indicate that a process identical to microhomology-driven single-strand annealing resolves L1 inversion intermediates.

Chromosome Inversion↗

PubMed2006

Insertional mutagenesis in C. elegans using the Drosophila transposon Mos1: a method for the rapid identification of mutated genes.

One benefit of the nematode Caenorhabditis elegans as a model system is the ease to conduct forward genetic screens and to isolate mutants with phenotypes of interest. However, identifying the mutated genes requires positional cloning, which can be laborious and time consuming. Insertional mutagenesis with a heterologous transposon bypasses the mapping steps and expedites the process of identifying the mutated genes. The Drosophila transposon Mos1 can be mobilized in the C. elegans germline to cause mutations. Mutagenic insertions are subsequently localized within the genome using inverse polymerase chain reaction. The mutagenicity of this technique is roughly one order of magnitude lower than chemical mutagens. However, the molecular identification of the mutated genes is extremely rapid. Therefore, before using Mos1-mediated mutagenesis, one must evaluate the trade-off between time spent screening for mutants vs time spent mapping and rescuing a mutation.

Animals↗

PubMed2003

Prior knowledge about display inversion in biological motion perception.

Display inversion severely impedes veridical perception of point-light biological motion (Pavlova and Sokolov, 2000 Perception & Psychophysics 62 889-899; Sumi, 1984 Perception 13 283-286). Here, by using a spontaneous-recognition paradigm, we ask whether prior information about display orientation improves biological motion perception. Participants were shown a set of 180 degrees inverted point-light stimuli depicting a human walker and quadrupeds (dogs). In experiment 1, one group of observers was not aware of the orientation of stimuli, whereas the other group was told beforehand that stimuli will be presented upside down. In experiment 2, independent groups of participants informed about stimulus orientation saw the same set of stimuli, in each of which either a moving or a static background line was inserted. The findings indicate that information about display inversion is insufficient for reliable recognition of inverted point-light biological motion. Instead, prior information facilitates display recognition only when it is complemented by additional contextual elements. It appears that visual impressions from inverted point-light stimuli remain impenetrable with respect to one's knowledge about display orientation. The origins of orientation specificity in biological motion perception are discussed in relation to the recent neuroimaging data obtained with point-light stimuli and fragmented Mooney faces.

Adolescent↗

PubMed1996

The herpes simplex virus type 1 UL6 protein is essential for cleavage and packaging but not for genomic inversion.

The HSV-1 genome is composed of two unique regions (UL and US) flanked by inverted repeats. During the course of DNA replication the two unique regions UL and US invert relative to one another. In this report we present evidence that cleavage is not necessary for genomic inversion to occur. We isolated and characterized a UL6::lacZ insertion mutant (hr74) that produces wild-type levels of replicating viral DNA but fails to cleave and package DNA. We demonstrate that this virus is still able to undergo genomic inversion. Furthermore we confirm that replicating DNA from cells infected with wild-type virus contains specific UL terminal but not US termini, whereas cells infected with the mutant hr74 do not contain either US or UL termini. This demonstrates that the specific UL ends found in replicating DNA are the result of the cleavage/packaging process.

Animals↗

PubMed2002

Transposition of DEH, a broad-host-range transposon flanked by ISPpu12, in Pseudomonas putida is associated with genomic rearrangements and dehalogenase gene silencing.

Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families. The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehR(I), was isolated from strain PP3 by using the TOL plasmid pWW0. DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehR(I) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria. Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P. putida PaW340. Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P. putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing. Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.

Bacterial Proteins↗

PubMed2003

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans.

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.

Amino Acid Sequence↗

PubMed1993

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.

Bacterial Proteins↗

PubMed1998

Insertional mutation by transposable element, L1, in the DMD gene results in X-linked dilated cardiomyopathy.

X-linked dilated cardiomyopathy (XLDCM) is a clinical phenotype of dystrophinopathy which is characterized by preferential myocardial involvement without any overt clinical signs of skeletal myopathy. To date, several mutations in the Duchenne muscular dystrophy gene, DMD , have been identified in patients with XLDCM, but a pathogenic correlation of these cardiospecific mutations in DMD with the XLDCM phenotype has remained to be elucidated. We report here the identification of a unique de novo L1 insertion in the muscle exon 1 in DMD in three XLDCM patients from two unrelated Japanese families. The insertion was a 5'-truncated form of human L1 inversely integrated in the 5'-untranslated region in the muscle exon 1, which affected the transcription or the stability of the muscle form of dystrophin transcripts but not that of the brain or Purkinje cell form, probably due to its unique site of integration. We speculate that this insertion of an L1 sequence in DMD is responsible for some of the population of Japanese patients with XLDCM.

5' Untranslated Regions↗

PubMed1998

Defective forms of cotton leaf curl virus DNA-A that have different combinations of sequence deletion, duplication, inversion and rearrangement.

Tobacco and tomato plants inoculated at least 9 months previously with a Pakistani isolate of cotton leaf curl virus (CLCuV-PK), a whitefly-transmitted geminivirus, contained substantial amounts of circular dsDNA molecules that were mostly about half the size of CLCuV-PK dsDNA-A. They appeared to be derived from CLCuV-PK DNA-A by various combinations of sequence deletion, duplication, inversion and rearrangement and, in a few instances, insertion of sequences of unknown origin. Each of ten tobacco plants contained a different predominant form of such a defective molecule; however, all the forms contained the intergenic region and part of the AC1 (Rep) gene. Some of the forms contained novel open reading frames and might have a role in the evolution of variant geminiviruses. The defective components were not detected at 3 months after the original culture of CLCuV-PK was transmitted by whiteflies (Bemisia tabaci) from cotton to tomato but were present after a further 6 months. They were transmitted, along with full-length DNA-A, between tobacco and tomato plants by grafting and by B. tabaci.

Animals↗