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PubMed1996

A purified mariner transposase is sufficient to mediate transposition in vitro.

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.

Animals↗
PubMed1996

Autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters.

Several classes of viruses are in use, or are being developed, as gene therapy vectors. Viruses with small genomes containing few essential genes have the advantage of requiring only simple complementation systems to allow packaging of foreign DNA, substituted for the entire viral coding sequences. Retroviruses and the dependent parvovirus AAV (adeno-associated virus) have been used in this way, and both possess an efficient integration mechanism which should allow long-term expression of transduced genes. In some situations, however, long-term persistence may be undesirable and there is a need for small, non-integrating viral vectors. Autonomous parvoviruses, such as LuIII, have potential as such vectors for short-term expression of therapeutic genes. We previously described recombinants of LuIII that transduced reporter genes, expressed using the viral constitutive promoter, P4. We have now generated several recombinants containing regulated promoters. A virus including a liver-specific enhancer directed 10- to 20-fold preferential expression of the luciferase reporter in transduced human hepatoma (HepG2) versus HeLa cells. In additional LuIII recombinants, the luciferase reporter was linked with chimeric promoters containing binding sequences for either the yeast GAL4 protein or the bacterial tetracycline repressor. Luciferase expression was strongly activated when these viruses were used to infect cells containing a cognate trans-activator (GAL4 or tTA, a tetracycline repressor fusion with VP16 of herpes simplex), introduced by transfection. The response to tTA could be abolished, or reduced in a graded manner, by exposure of the infected cells to tetracycline. Further results suggested that an increase in basal expression, apparently mediated by the viral left terminal inverted repeat, could be minimized by interposing polyadenylation signals between this sequence and the promoter. These results confirm that appropriate transcriptional regulation can be achieved for genes transduced by an autonomous parvovirus vector. Such vectors therefore show promise for the delivery of therapeutic genes in situations requiring cell-specific, short-term expression, eg in targeting suicide genes for ablation of cancer cells.

Cell Line↗
PubMed1996

[Sequencing of right terminal fragment of canine adenovirus type 1 vaccine strain DNA].

Current studies show that adenoviruses are more advantageous as expression vectors than other viruses. Canine adenovirus type 1 (CAV-1) vaccine strain, CLL (cannaught laboratory limited), as vectors will play more important role for the prophylaxis of zoonosis than human adenoviruses. Studies on molecular biology of CAV-1 or CLL are necessary for construction of vectors of CLL. In this paper, the right terminal of CLL DNA cleaved by Hind II was sequenced. Authors found that it contained a 238 bp inverted terminal repeats (ITR). There are three copies of 40 bp short repeat sequences in the ITR. According to the DNA structural model of human adenovirus type 2, the 40 bp short repeats are the sites that are combined with nuclear factor 1 (NF-1) that is an initiation factor of DNA replication of virus, so they are essential structure of CLL DNA.

Adenoviruses, Canine↗
PubMed1997

BARE-ID, a representative of a family of BARE-like elements of the barley genome.

In our search for transposable elements in barley, Hordeum vulgare, we have isolated and cloned two BamHI-fragments of 4.7 and 4.2 kb in length containing very abundant DNA sequences. The 4.7 kb fragment is homologous to the extended region, including more than half of the 5'-LTR and some part of the coding domain of BARE-1, a member of copia-like retrotransposon family of barley. The 4.2 kb fragment, bearing homology to BARE-1 and the WIS-2 family isolated from wheat, is unique among studied retroelements of cereals because it consists of two inverted parts, each containing homology to the LTR and UTL of BARE-1. Functional motifs for reverse transcription, two TATA-boxes and two primer-binding sites, were found within the LTRs. The element contained within this fragment was generated by significant rearrangement of a BARE-like retrotransposon, which included inversion of the extended 5'-terminal region and deletion of the internal domain. Therefore this element is named BARE-ID (BARE-inverted, deleted). A family of BARE-like elements is amplified in the H. vulgare genome compared with wild barley species. The terminal inverted repeat of BARE-ID was used as a probe for examination of evolutionary diversity within genus Hordeum. Our data are basically in agreement with the modern classification system. However, they do not support the combination of H. vulgare and H. bulbosum into one group with the same type of genome. New data concerning the possible origin of the polyploid species, H. secalinum, confirm that retrotransposons are a useful tool for phylogenetic studies.

Base Sequence↗
PubMed1998

An active Ac-like transposable element in teleost fish.

The i4/i4 genotype of the medakafish, Oryzias latipes, exhibits a quasi albino phenotype due to insertion of a novel transposable element, Tol2, into the tyrosinase gene. Tol2 is 4681 bp in length, has short inverted terminal repeats, and contains four ORFs with the potential to encode a transposase protein. Excision activity of the element has been detected by PCR analysis. Genomic Southern of the Tol2 element revealed that about 20 copies are present in the diploid genome. Dot-matrix comparisons of amino acid sequences of ORFs show relatively high similarity with transposases from Ac of maize, hobo of Drosophila, and Tam3 of snapdragon, which are all active transposable elements. Tol2 is thus concluded to be an active Ac-like transposable element probably encoding a transposase protein. It should therefore find application as a unique material for establishing a gene tagging system in fish.

Journal Article↗
PubMed1985

Nucleotide sequence of the S-1 mitochondrial DNA from the S cytoplasm of maize.

Mitochondria from the S male-sterile cytoplasm of maize contain unique DNA-protein complexes, designated S-1 and S-2. These complexes consist of double-stranded linear DNAs with proteins covalently attached to the 5' termini. To learn more about these unusual DNAs we have determined the complete nucleotide sequence of the S-1 DNA molecule (6397 bp). The sequence of S-2 has been previously determined. S-1 and S-2 are structurally similar and contain 1.7kb of sequence homology. S-1 is terminated by exact 208-bp inverted repeats that are identical with the terminal inverted repeats of S-2. S-1 and S-2 also contain a 1462-bp region of nearly perfect homology, which includes one of the terminal inverted repeats. The homology between the two molecules may be maintained, in part, by homologous recombination. S-1 has three long unidentified open reading frames, URF2 (1017 bp), URF3 (2787 bp) and URF4 (768 bp). URF2 occurs in the 1462-bp region of homology and is identical in length and location in both S-1 and S-2. Based on their structural organization and their viral-like characteristics, we propose that S-1 and S-2 code for functions involved with their maintenance and replication.

Journal Article↗
PubMed2005

DIRS-1 and the other tyrosine recombinase retrotransposons.

DIRS-1 is a retroelement from the slime mold Dictyostelium discoideum. Until recently only two related retrotransposons had been described: PAT from the nematode Panagrellus redivivus and Prt1 from the zygomycete fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these three elements suggested that they were closely related to each other and more distantly related to the Ty3/gypsy Long Terminal Repeat (LTR) retroelements. They have several unusual structural features that distinguish them from typical LTR elements. For instance, they each encode a tyrosine recombinase (YR), but not a DDE-type integrase or an aspartic protease. Although the DIRS-1-related elements are bordered by terminal repeats these differ from typical LTRs in a number of ways. In DIRS-1, for example, the terminal repeats are inverted (complementary), non-identical in sequence, and the outer edges of the terminal sequences are repeated (adjacent to each other) in the internal region. PAT has so-called "split" direct repeats in which the unrelated terminal sequences appear as direct repeats adjacent to each other in the internal region. The only repetition displayed by Prt1 is the presence of short inverted terminal repeats, but the sequenced copy of this element is believed to be a truncated version of an element with a structure resembling DIRS-1. The unusual structure of the terminal repeats of the DIRS1-like elements appears to be related to their replication via free circular intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. In recognition of these important distinctions it is proposed that the retrotransposons that encode tyrosine recombinases be called the tyrosine recombinase (or YR) retrotransposons. Recently a large number of additional YR retrotransposons have been described, including elements from fungi (zygomycetes and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish and amphibia, while remnants of elements related to DIRS-1 occur in the human genome. The complete set of YR retrotransposons can be divided into two major groups, the DIRS elements and the Ngaro elements, the two groups forming distinct clades on phylogenetic trees based on alignments of RT/RH and recombinase sequences, and also having some structural distinctions. A third group of transposable elements, which we call Cryptons, also carry tyrosine recombinases. These elements do not encode a reverse transcriptase and so are believed to be DNA transposons not retrotransposons. They have been detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. Sequence comparisons suggest that the Crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a Crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon.

Amino Acid Sequence↗
PubMed1999

A mutation in the flanking 5'-TA-3' dinucleotide prevents excision of an internal eliminated sequence from the Paramecium tetraurelia genome.

The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5'-TA-3' dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5'-TA-3' and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5'-TA-3' dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.

Animals↗
PubMed1987

Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.

Amino Acid Sequence↗
PubMed2002

A p5 integration efficiency element mediates Rep-dependent integration into AAVS1 at chromosome 19.

Adeno-associated virus (AAV) undergoes site-specific integration into human chromosome 19 through a deletion-substitution mechanism at the well characterized AAVS1 site. We have shown previously that a cis element within the left end of the AAV genome enhances the efficiency of Rep-mediated site-specific integration into chromosome 19 when present in inverted terminal repeat-containing recombinant AAV (rAAV) plasmids. We now demonstrate that a 138-bp cis element, the p5 integration efficiency element (p5IEE), mediates efficient integration. The p5IEE is not only required for efficient site-specific integration, it is also sufficient. Integration mediated by the p5IEE occurs in the absence of the AAV inverted terminal-repeat elements. The data presented in this study demonstrate that the p5IEE is a multifunctional element, serving as the highly regulatable Rep promoter and the primary substrate for targeted integration.

Base Sequence↗
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↗
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↗