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PubMed1982

DNA sequence analysis of an immediate-early gene region of the herpes simplex virus type 1 genome (map coordinates 0.950 to 0.978).

The nucleotide sequence of 4 kilobases of DNA from within the short region of the genome of herpes simplex virus type 1 has been determined. This portion of DNA contains the junctions of the terminal and inverted repeat sequence components with the unique sequence component and the 5'-regions of the genes which encode the Vmw 12, Vmw 68 and Vmw 175 immediate-early polypeptides. The transcription and translation initiation sites of all three genes and the 5' and 3' boundaries of the Vmw 12 and Vmw 68 gene introns have been localized on the DNA sequence and shown to be flanked by sequences which resemble those found in similar positions in other eukaryotic genes. For the Vmw 12 and Vmw 68 genes the promoters, the 5'-non-coding regions of the mRNAs, and the introns lie within the terminal and internal inverted repeats respectively while the polypeptide-coding regions lie in the short unique component. The introns consist largely of tandemly reiterated copies of a 22-nucleotide sequence: the Vmw 12 gene intron contains seven of these and the Vmw 68 gene intron five. The Vmw 175 gene is located entirely within the short repeats, of which those regions sequenced here have the extremely high G + C content of 78%, in marked contrast to the value of 66% obtained for the adjacent region of the unique sequence component. Prediction of the complete amino acid sequence of the Vmw 12 polypeptide, accounting for a mol. wt. of 9830, and of the first 523 amino-terminal amino acids of the Vmw 175 polypeptide has been made from the DNA sequence. The polypeptide-coding region of the Vmw 175 gene has an average G + C content of 80% but nevertheless specifies a wide range of amino acid types because of the maximal assignment of G and C residues to colon third base positions.

Amino Acid Sequence↗
PubMed1996

Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm6, a small transposable element that belongs to the IS3 family.

The insertion sequence ISRm6 is a small transposable element identified in Rhizobium meliloti strain GR4 by sequence analysis. Two copies of this IS element were found in strain GR4, one of them is linked to the nfe genes located on plasmid pRmeGR4b. ISRm6 seems to be widespread in R. meliloti. Data suggest that ISRm6 is active in transposition at an estimated frequency of 2 x 10(-5) per generation per cell in strain GR4. This 1269-bp element carries 27/26-bp terminal imperfect inverted repeats with six mismatches and a direct target site duplication of 4 bp. The IR terminate with the dinucleotide 5'-TG as all the members of the IS3 family. In addition, as other IS belonging to the IS3 family, ISRm6 carries two open reading frames (ORFA and ORFB) with a characteristic translational frame-shifting window in the overlapping region. Furthermore, ISRm6 putative transposase contains the triad of amino acids called DDE motif. Comparison of the ISRm6 DNA sequence and the putative proteins encoded with sequences derived from the EMBL, GenBank, PIR and Swissprot databases showed significant similarity to IS that belongs to the IS3 family with a highest homology to a subclass containing IS476 from Xanthomonas campestris, IS407 from Burkholderia cepacia, and ISR1 from Rhizobium lupini.

Base Sequence↗
PubMed1994

Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus.

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.

Amino Acid Sequence↗
PubMed1992

Inverted repeats in the long-terminal repeats of the wheat retrotransposon Wis 2-1A.

The wheat insertion sequence Wis 2-1A possesses all the structural features characteristic of retrotransposons. Its long-terminal repeats (LTRs) are unusually long (1,755 bp) compared with those of other retrotransposons. Sequence analysis revealed that they differ from each other by only six point mutations. They contain a few tandem direct repeats, which could be explained by slippage mechanisms during replication. Almost half (44%) of the length of the LTRs is occupied by hairpin structures, which may relate to their large size. Possible origins of these inverted repeats are proposed, including the insertion and imprecise excision of transposable elements and errors when the DNA replication intermediate switches RNA template during retrotransposon replication.

Base Sequence↗
PubMed2006

Self-splicing of a group IIC intron: 5' exon recognition and alternative 5' splicing events implicate the stem-loop motif of a transcriptional terminator.

Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interestingly, additional unexpected products were formed that were highly dependent on ionic conditions. These products were determined to represent alternative splicing events at the 5' junction and cleavages throughout the RNA transcript. The alternative splicing and cleavage events occurred at cryptic splice sites containing stem-loop and IBS1 motifs, suggesting that the 5' exon is recognized by both elements. These results provide the first example of a group II intron that uses 5' splice sites nonadjacent to the ribozyme structure. Furthermore, the data suggest that IIC introns differ from IIA and IIB introns with respect to 5' exon definition, and that the terminator stem-loop substitutes in part for the missing IBS2-EBS2 (intron and exon binding sites 2) interaction.

Alternative Splicing↗
PubMed2002

Cloning and characterization of Acmar1, a mariner-like element in the asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita).

A mariner-like element was cloned from the genome of the Asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita). The (composite) clone, named Acmar1, was 1,378 bp long, and encoded 336 amino acids corresponding to a transposase-like putative polypeptide in a single open reading frame. The D,D(34)D motif, the catalytic domain of the mariner transposase, was present, although there was a deletion of five amino acid residues within it as compared with the active transposase in Drosophila mauritiana. Nineteen-bp-long imperfect inverted terminal repeat-like sequences flanked by TA dinucleotides, the typical target site for mariner insertion, were observed. Southern blot analysis using a fragment covering two-thirds of the Acmar1 transposase coding sequence as a probe indicated the presence of multiple Acmar1-like elements in the genome. Maximum-parsimony phylogenetic analysis based on the transposase amino acid sequences of insect mariner-like elements revealed that Acmar1 is a member of the mellifera subfamily.

Amino Acid Sequence↗
PubMed1995

The isolation of Ant1, a transposable element from Aspergillus niger.

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 bp in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3' coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.

Amino Acid Sequence↗
PubMed2006

Covalent attachment of multifunctional chimeric terminal proteins to 5' DNA ends: A potential new strategy for assembly of synthetic therapeutic gene vectors.

The generation of synthetic therapeutic gene vectors requires the coupling of DNA to transfer-promoting peptides including cellular receptor ligands, protein transduction domains, hydrophobic peptides for attachment to lipid membranes, nuclear localisation signals, cytoskeleton attachment motifs, nuclear matrix association elements and immune evasion moieties. Existing methods of peptide-DNA joining often interfere with transgene expression and, therefore, are inadequate for production of effective therapeutic vector complexes, particularly destined for gene delivery in the challenging environment in vivo. However, there is a natural mechanism for rigid coupling of polypeptides with DNA. Some bacterial and eukaryotic linear plasmids, adenoviruses and a number of bacteriophages including phi29 of Bacillus subtilis and PRD1 of Escherichia coli use terminal proteins covalently bound to 5' DNA ends to prime replication. Inverted terminal DNA repeats, normally short DNA sequences, contain all the sequences required in cis for the covalent coupling reaction. The complex of the terminal protein, DNA polymerase and some known auxiliary proteins supplies sufficient trans-functions, thus enabling simple linking of the terminal proteins to DNA in vitro. We hypothesise that chimeric fusion proteins, constructed on the basis of terminal proteins of adenoviruses, linear plasmids or bacteriophages with protein-primed replication, can on the one hand retain the ability to bind covalently 5' DNA termini in conditions established previously for protein-primed replication in vitro, and on the other hand confer gene transfer facilitating properties and enhanced longevity of efficient transgene expression. Terminal localisation of the chimeric proteins can ensure that they do not interfere with transgene transcription. At the same time a covalent bond between polypeptide and DNA can provide rigid coupling ensuring their stable association en route to nuclei. Bound to 5'-ends of the delivered DNA, terminal protein-based chimeras could also protect the vector DNA from 5'-3' and possibly 3'-5' exonuclease attack, thus limiting its intracellular degradation and increasing longevity of transgene expression. Our hypothesis can be tested by measuring the gene transfer efficiency of the novel complexes containing linear DNA fragments with covalently linked multifunctional chimeric terminal proteins, using previously described synthetic gene vectors as standards.

5' Untranslated Regions↗
PubMed2002

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors.

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Adenoviridae↗
PubMed1994

Linear DNA plasmids from Pichia etchellsii, Debaryomyces hansenii and Wingea robertsiae.

Linear DNA plasmids were found in the following yeasts: four strains of Kluyveromyces lactis, one of Debaryomyces hansenii, one of Wingea robertsiae and four of Pichia etchellsii. In each case, the plasmids were present as a pair of DNA molecules of different sizes. The plasmids of K. lactis strains were associated with a killer activity and their structure was similar to the known killer plasmids pGKL1 and 2. The plasmids from the other three species were different from pGKL plasmids and showed no killer activity against the yeast species tested so far. In all cases, the linear molecules possessed terminal (probably inverted) repeats and their 5' ends had a protected structure insensitive to lambda exonuclease, while the 3' ends were accessible to exonuclease III. All these strains could be efficiently cured of the plasmids by ultraviolet irradiation. The plasmids from D. hansenii (pDH1A and B) and from W. robertsiae (pWR1A and B) shared related sequences with some of the K. lactis killer plasmid genes (encoding the supposed DNA polymerases, RNA polymerase and the chitinase), suggesting related genome organization of these plasmids. The pair of plasmids from P. etchellsii (pPE1A and B) appear to be a distantly related member of the group. This pair showed no sequence homology with other plasmids, except weak homology with the putative RNA polymerase gene of pGKL2. None of the plasmids contained the sequences homologous to ORF3 and ORF4 of pGKL1 encoding the toxin resistance determinant and the toxin gamma subunit, respectively.

DNA, Fungal↗
PubMed1995

IS1500, an IS3-like element from Leptospira interrogans.

Copies of an insertion-sequence (IS)-like element were isolated from two closely related serovars of Leptospira interrogans sensu stricto. Nucleotide sequence analysis of the 1236 bp element showed a characteristic IS structure with terminal imperfect inverted repeats (IRs) flanking a 1159 bp central region. This element was designated IS1500. Four open reading frames (orfA-orfD) were found in the central 'unique' region of IS1500. Similarities were detected between ORFA and ORFB and the putative transposases from members of the IS3 family of transposable elements. IS1500 or IS1500-like sequences were also detected in all other pathogenic leptospiral serovars, but not in the saprophytic species L. biflexa. Differences in IS1500 copy numbers in members of the same species suggest that this element can transpose. Physical mapping of IS1500 insertions in L. interrogans serovars icterohaemorrhagiae and pomona showed insertions were only on the large chromosomal replicon. The location of some IS1500 insertions coincides with regions of the genome that have undergone large rearrangements.

Amino Acid Sequence↗
PubMed1998

A new insertion sequence from Sinorhizobium meliloti with homology to IS1357 from Methylobacterium sp. and IS1452 from Acetobacter pasteurianus.

The insertion sequence ISRm8 was identified by sequence analysis of the cryptic plasmid pRmeGR4b of Sinorhizobium meliloti GR4. ISRm8 is 1451 bp in length and carries 22/24-bp terminal imperfect inverted repeats with seven mismatches and a direct target site duplication of 3 bp. ISRm8 carries a unique open reading frame whose putative protein showed significant similarity to the insertion sequences IS1357 and IS1452, isolated from Methylobacterium sp. and Acetobacter pasteurianus, respectively. Two copies of this IS element were found in strain GR4; one of them is linked to plasmid pRmeGR4b, whereas the other is localized out of the non-pSym plasmids. In S. meliloti field populations ISRm8 shows a limited distribution (50% of the strains tested carry the IS element), with a copy number ranging from 1 to 6.

Acetobacter↗
PubMed1993

IS870 requires a 5'-CTAG-3' target sequence to generate the stop codon for its large ORF1.

The TB regions of the Agrobacterium vitis octopine/cucumopine Ti plasmids constitute a family of related structures. All contain a bacterial insertion element downstream of the TB-iaaM gene, IS870.1. Whereas 43 isolates with octopine/cucumopine Ti plasmids carry only one IS870 copy, strain Ag57 carries a second copy (IS870.2) 3.9 kb to the right of IS870.1 and part of the same TB region. Two other octopine/cucumopine strains carry an IS870 copy on their chromosome (IS870.3). A study of the unmodified insertion sites of IS870.2 and IS870.3, cloned from closely related strains, enabled us to delimit the IS870 elements. IS870 has a size of 1,152 bp and is terminated by inverted repeats. It contains a large open reading frame without a stop codon. However, a stop codon is generated by insertion into the target sequence 5'-CTAG-3'. IS870 is related to five other insertion sequence elements. For two of these, the stop codon of the largest open reading frame is also created by insertion into a CTAG target site.

Amino Acid Sequence↗
PubMed2002

Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis.

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

Adenoviruses, Human↗
PubMed2002

Kinetics and frequency of adeno-associated virus site-specific integration into human chromosome 19 monitored by quantitative real-time PCR.

Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.

Chromosomes, Human, Pair 19↗
PubMed1990

A retrotransposable element from the mosquito Anopheles gambiae .

A family of middle repetitive elements from the African malaria vector Anopheles gambiae is described. Approximately 100 copies of the element, designated T1Ag, are dispersed in the genome. Full-length elements are 4.6 kilobase pairs in length, but truncation of the 5' end is common. Nucleotide sequences of one full-length, two 5'-truncated, and two 5' ends of T1Ag elements were determined and aligned to define a consensus sequence. Sequence analysis revealed two long, overlapping open reading frames followed by a polyadenylation signal, AATAAA, and a tail consisting of tandem repetitions of the motif TGAAA. No direct or inverted long terminal repeats (LTRs) were detected. The first open reading frame, 442 amino acids in length, includes a domain resembling that of nucleic acid-binding proteins. The second open reading frame, 975 amino acids long, resembles the reverse transcriptases of a category of retrotransposable elements without LTRs, variously termed class II retrotransposons, class III elements or non-LTR retrotransposons. Similarity at the sequence and structural levels places T1Ag in this category.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of the complex sex-chromosome heterochromatin in the rodent Microtus chrotorrhinus.

The sex chromosomes of Microtus chrotorrhinus are unusually large compared to those of other mammals, comprising about 20% of the karyotype and containing substantial amounts of constitutive heterochromatin. Previous studies have described two highly repeated DNA families (MSAT-160 and MSAT-2570) that localize to this heterochromatin (Modi, 1992, 1993c). The present report describes a third satellite DNA family (termed MSAT-21) in M. chrotorrhinus that is also located in the sex heterochromatin. This repeat consists of diverged copies (average similarity = 75%) of a tandemly repeated 21-mer. Southern blotting of MSAT-21 revealed that although some higher order (5-20 kb) repeats do exist, none has spread throughout an appreciable portion of the genome. Pulsed field gel experiments indicated that most of the larger arrays (50-700 kb) of all three satellite families are distributed across numerous size classes, suggesting that the three repeats are interspersed with one another in this heterochromatin. Analysis of a boundary between MSAT-21 and MSAT-160 showed that the junction monomers of each satellite are intact and that a pentanucleotide has apparently been transferred from MSAT-21 to MSAT-160 via recombination. Sequence comparisons of MSAT-160 with another rodent satellite and with the U3 region of the Rous sarcoma virus (RSV) long terminal repeat identified inverted repeats and similarities with viral enhancer domains in the rodent sequences. Additionally, the MSAT-21 consensus was found to be similar to the R region of RSV, suggesting a retroviral ancestor for these rodent repeated DNA families.

Animals↗
PubMed1994

Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae.

Co-ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins. These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes. The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR. Primer extension and gene fusion analyses identified a ToxR-regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription. Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT. Possible mechanisms of ToxR-dependent toxT expression are discussed.

Bacterial Proteins↗