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PubMed2002

Development of multiple cloning site cis-vectors for recombinant adeno-associated virus production.

Recombinant adeno-associated virus (rAAV) has become a very popular gene therapy vector in the past several years. A cis-plasmid is used to generate the rAAV stocks. In this plasmid, the entire expression cassette is incorporated between two AAV inverted terminal repeats. The construction of cis-plasmid has been problematic because of the high-frequency recombination of the viral inverted terminal repeats. Here we describe the design and construction of several multiple cloning site cis-plasmids that are driven by five different promoters, including the ubiquitous cytomegalovirus enhancer/chicken beta-actin (CAG), cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus 40 (SV40), and a muscle-specific promoter (CK6). The application of these multiple cloning site cis-plasmids improves the cloning efficiency. As an example of the utilization of these multiple cloning site vectors, the prokaryotic beta-galactosidase cDNA was cloned in the multiple cloning site cis-plasmids. High-level rAAV-mediated beta-galactosidase expression was achieved in HeLa cells from CAG, CMV, RSV and SV40 promoters, respectively, but notfrom the CK6 promoter. In vivo application in the adult mdx mouse (mouse model for Duchenne muscular dystrophy) muscle revealed efficient transgene expression from CMV and CK6 promoters, followed by CAG and RSV promoters. The SV40 promoter was the least efficient.

Animals↗
PubMed1994

A dispersed family of repetitive DNA sequences exhibits characteristics of a transposable element in the genus Lycopersicon.

A segment of DNA 5' to the transcribed region of an auxin-regulated gene, ARPI, from Lycopersicon esculentum Mill. cv. VFN8 contains a sequence with the structural characteristics of a transposable element. The putative element (Lyt1) is 1340 bp long, has terminal inverted repeats of approximately 235 bp and is flanked by 9-bp direct repeats. Lyt1 has a structure similar to the Robertson's Mutator (Mu) family from maize. The terminal inverted repeats are 80% AT-rich, are 96.6% identical, and define a larger family of repetitive elements. Southern analysis and genomic dot-blot reconstructions detected at least 41 copies of Lyt1-hybridizing sequences in red-fruited Lycopersicon spp. (L. esculentum, L. pimpinellifolium and L. cheesmanii), and 2-8 copies in the green-fruited species (L. hirsutum, L. pennellii, L. peruvianum, L. chilense and L. chmielewskii). There were two to four copies in the Solanum spp. closely allied with the genus Lycopersicon (S. lycopersicoides, S. ochranthum and S. juglandifolium), while the more distantly related Solanum spp. showed little (one to two copies in S. tuberosum) to no (S. quitoense) detectable hybridization under stringent conditions. Linkage analysis in the F2 progeny of a cross between L. esculentum and L. cheesmanii indicated that at least six loci that hybridize to the Lyt1 sequence are dispersed in the genome. Polymerase chain reaction and Southern analyses revealed that some red-fruited accessions and L. chmielewskii lacked Lyt1 5' to the transcribed region of ARPI. Subsequent sequence analysis indicated that only one copy of the 9-bp direct repeat (target site) was present, suggesting that transposition of the element into the ARPI gene occurred after the divergence of the red-fruited and green-fruited Lycopersicon species.

Base Sequence↗
PubMed1991

Tc4, a Caenorhabditis elegans transposable element with an unusual fold-back structure.

We have identified and characterized a family of transposable elements in the nematode Caenorhabditis elegans. The Tc4 transposable element family is present at about 20 copies per haploid genome in the C. elegans Bristol and Bergerac strains. Although Tc4 transposition events have not been observed in these wild-type strains, we have identified Tc4 transposition events in the mut-2 mutant strain TR679, in which the elements Tc1 and Tc3 also transpose at a higher frequency than in the wild type. We determined the sequence of one Tc4 element. This 1.6-kilobase element contains almost perfect inverted terminal repeats of 774 base pairs (bp) with a 57-bp unique internal sequence. Tc4 is a fold-back element, but its long inverted terminal repeats, unlike those of the fold-back elements of other organisms, do not consist of multiple short repeats. In the two cases studied, Tc4 insertion resulted in duplication of a TNA trinucleotide target site. The family of Tc4 elements differs from other C. elegans transposable element families in structure, degree of structural heterogeneity, and target-site specificity.

Animals↗
PubMed1983

Discrete regions of sequence homology between cloned rodent VL30 genetic elements and AKV-related MuLV provirus genomes.

Southern blot analyses using reduced stringency hybridization conditions have been employed to search for sequence homologies between rodent VL30 genes and murine leukemia virus (MuLV) proviruses. These constitute two classes of transposon-like elements previously believed to be genetically unrelated. Our results demonstrate that cloned representatives of both ecotropic and xenotropic-like proviruses share discrete regions of sequence homology with VL30 genes of both rat and mouse origin. These regions of homology exist in both 3' and 5' halves of the MuLV genome but do not include extensive portions of the long terminal repeat (LTR) or a 0.4 Kbp segment of the env gene specific for recently acquired ecotropic-type MuLV proviruses. DNA sequencing, however, revealed that the short inverted terminal repeat sequence of MuLV proviral LTRs is almost perfectly conserved at the terminus of an integrated mouse VL30 gene. These results suggest that recombination events with rodent VL30-type sequences occurred during early MuLV evolution. The strong conservation of the inverted terminal repeat sequence may reflect a common integration mechanism for VL30 elements and MuLV proviruses.

Animals↗
PubMed2000

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.

Arabidopsis↗
PubMed1987

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family.

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.

Animals↗
PubMed2000

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster.

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.

Animals↗
PubMed1987

Isolation and characterization of IS elements repeated in the bacterial chromosome.

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.

Chromosomes, Bacterial↗
PubMed1992

Presence of a member of the Tc1-like transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptatretus stouti.

Molecular cloning of the vasotocin gene of a cyclostome, the Pacific hagfish Eptatretus stouti, reveals, in contrast to other known members of the vertebrate vasopressin/oxytocin hormone gene family, an unusual exon-intron organization. Although the location of three exons and two introns is conserved, an additional intron is present 5' of the coding region of the hagfish gene. The third intron, which is greater than 14 kilobase pairs in size, contains on the opposite DNA strand to that encoding vasotocin an open reading frame exhibiting striking similarity to the putative transposase of Tc1-like nonretroviral mobile genetic DNA elements, so far reported only from nematodes and Drosophila. The hagfish element, called Tes1, is flanked by inverted terminal repeats representing an example of the existence of a typical inverted terminal-repeat transposon within vertebrates. The presence of Tc1-like elements in nematodes, Drosophila, and cyclostomes indicates that these genetic elements have a much broader phylogenetic distribution than hitherto expected.

Amino Acid Sequence↗
PubMed1997

Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome.

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.

Adenoviruses, Human↗
PubMed1995

Large deletions in antigenic variants of the chlorella virus PBCV-1.

Four spontaneously derived, antigenic variants of chlorella virus PBCV-1 contained 27- to 37-kb deletions in the left end of the 330-kb genome. Two of the mutants, which were serologically identical, had deletions that began from map position 4.9 or 16 and ended at position 42.2 kb. In total, the two deleted regions encoded 28 putative functional open reading frames (ORFs); these deletions probably arose from homologous recombination. The other two mutants, which were serologically identical but distinct from the first two mutants, lacked the entire left terminal 37 kb of the PBCV-1 genome, including an identical 2.2-kb inverted terminal repeat region present at both ends of the wild-type genome. The deleted left end region was replaced by the transposition of an inverted 7.7- or 18.5-kb copy of the right end of the PBCV-1 genome. The region deleted in these two viruses encoded 26 single-copy ORFs, of which 23 were common to those deleted in the first two mutant viruses. The junctions of the deletions/transpositions probably arose from nonhomologous recombination. Taken together, the results indicate that 40.1 kb of single-copy DNA encoding 31 ORFs at the left end of the genome are unnecessary for PBCV-1 replication in Chlorella strain NC64A in the laboratory. The results also indicate that the size of the inverted terminal repeat region in this virus can be highly variable and that the PBCV-1 DNA packaging process tolerates large changes in genome size.

Antigenic Variation↗
PubMed1999

The Queensland fruit fly, Bactrocera tryoni, contains multiple members of the hAT family of transposable elements.

Members of the hAT transposable element family are mobile in non-host insect species and have been used as transformation vectors in some of these species. We report that the Queensland fruit fly, Bactrocera tryoni, contains at least two types of insect hAT elements called Homer and a Homer-like element (HLE). The Homer element is 3789 bp in size and contains 12-bp imperfect inverted terminal repeats. The Homer element contains a long open reading frame (ORF) that encodes a putative transposase. Three different copies of this long ORF were recovered from the B. tryoni genome and, upon transcription and translation in an in vitro system, all produced transposase. The HLE is an incomplete element since no 3' inverted terminal repeat (ITR) was found. Homer and the HLE are as related to one another as either is to the other insect hAT elements such as Hermes, hobo, hermit and hopper. The structure and distribution of these two Homer elements is described.

Amino Acid Sequence↗
PubMed2001

The Arabidopsis TAG1 transposase has an N-terminal zinc finger DNA binding domain that recognizes distinct subterminal motifs.

The in vitro DNA binding activity of the Arabidopsis Tag1 transposase (TAG1) was characterized to determine the mechanism of DNA recognition. In addition to terminal inverted repeats, the Tag1 element contains four different subterminal repeats that flank a transcribed region encoding a 729-amino acid protein. A single site-specific DNA binding domain is located near the N terminus of TAG1, between residues 21 and 133. This domain binds specifically to the AAACCC and TGACCC subterminal repeats, found near the 5' and 3' ends of the element, respectively. The ACCC sequence within these repeats is critical for recognition because mutations at positions 3, 5, and 6 abolished binding, yet the first two bases also are important because substitutions at these positions decreased binding by up to 90%. Weak interaction also occurs with the terminal inverted repeats, but no binding was observed to the other two 3' subterminal repeat regions. Sequence analysis of the TAG1 DNA binding domain revealed a C(2)HC zinc finger motif. Tests for metal dependence showed that DNA binding activity was inhibited by divalent metal chelators and greatly enhanced by zinc. Furthermore, mutation of each cysteine residue predicted to be a metal ligand in the C(2)HC motif abolished DNA binding. Together, these data show that the DNA binding domain of TAG1 specifically binds to distinct subterminal repeats and contains a zinc finger.

Amino Acid Sequence↗
PubMed2002

Spontaneous mobilization of integrated recombinant adenoassociated virus in a cell culture model of virus latency.

A cell line containing integrated recombinant adenoassociated virus (AAV) was investigated for spontaneous mobilization of vector sequence. Detection of these rare events was facilitated by using a vector design that allowed the circular rescue product (cAAV) to be individually scored by bacterial transformation. Restriction and sequence analysis of captured clones revealed five highly ordered classes of cAAV, each of which contained a defined segment of the integrated vector locus. A common feature of all cAAV classes was the presence of a modified inverted terminal repeat that joined the ends of the liberated sequence. Assembly of extrachromosomal vector genomes was accompanied by deletions in the integration locus that could be mapped to one of the five cAAV classes, suggesting an excision-type mechanism. We propose that the spontaneous deletion and mobilization of vector sequence from the recombinant adenoassociated virus (rAAV) integration locus is mediated by a recombination event between the inverted terminal repeats that define the boundaries of the individual genome subunits.

Cell Line↗
PubMed1999

IS1631 occurrence in Bradyrhizobium japonicum highly reiterated sequence-possessing strains with high copy numbers of repeated sequences RSalpha and RSbeta.

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSalpha and RSbeta (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845-1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSalpha or RSbeta. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.

Amino Acid Sequence↗
PubMed1995

Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.

Base Sequence↗
PubMed2000

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

Actins↗
PubMed1988

Characterization of the origins of replication of bacteriophage phi 29 DNA.

The origins of replication of phi 29 DNA have been studied by analyzing the activity as templates in the phi 29 in vitro replication system of E. coli recombinant plasmids and M13 derivatives containing phi 29 DNA terminal sequences. Plasmid pITR, containing the 6 bp long inverted terminal repeat of phi 29 DNA, was shown to be essentially inactive. The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right phi 29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the mutagenesis at these sequences was carried out. Changes of the second or third A into a C completely abolished the template activity. In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat. The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.

Bacteriophages↗