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PubMed1994

Transcriptional activation of flanking sequences by Tn1000 insertion.

The carotenoid biosynthesis operon of Erwinia herbicola Eho13 consists of five genes, which are organized in the order crtE-crtX-crtY-crtI-crtB. These genes, with the exception of crtX, encode functions of beta-carotene biosynthesis and give an orange-coloured phenotype in Escherichia coli. Since crtX is not involved in the biosynthesis of beta-carotene, deletion of this gene does not alter the phenotype of pigmented cells. On the other hand, insertion of Tn1000 into crtX or into the upstream untranslated region of the operon resulted in a light-yellow, rather than an unpigmented phenotype, indicating that Tn1000 does not exert a strong polar effect when inserted in this operon. RNA analysis revealed that the sequence downstream from the insertion site was transcribed at a low level. Primer extension showed that the "-35"-like sequence in the terminal inverted repeats was not responsible for the transcription of the downstream sequence. Furthermore, primer extension and polymerase chain reaction (PCR) studies revealed that RNA transcribed from the promoters inside of Tn1000 was extended through the terminal inverted repeats into the adjacent sequences. In addition Tn1000, in either orientation, was able to generate fusion transcripts when placed upstream of a promoter-less tetracycline-resistance gene and resulted in cells resistant to the drug. These results showed that Tn1000 insertion transcriptionally activates the DNA sequences adjacent to the transposon.

Base 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↗
PubMed1988

Molecular characterization of suppressor-mutator (Spm)-induced mutations at the bronze-1 locus in maize: the bz-m13 alleles.

The bz-m13 allele of maize contains a defective Suppressor-mutator (dSpm) transposable element and gives rise to a variety of stable and unstable derivatives in the presence of an autonomous Suppressor-mutator (Spm) element. The dSpm-13 element of bz-m13 consists of 2,241 base pairs (bp) and is located within the second exon of the bronze-1 (bz) gene. A number of the stable derivatives, both functional and nonfunctional, derived from bz-m13 were characterized molecularly. Results from genomic DNA blotting experiments indicate that the dSpm-13 element had excised from the locus in each stable derivative analyzed. The unstable derivatives bz-m13CS9 and bz-m13CS6 contain dSpm elements in the same position and orientation as the dSpm-13 element, but they differ in the length of the element. The dSpm-13CS9 element is 902 bp and arose via a deletion between two 5-bp direct repeats within the dSpm-13 element. The dSpm-13CS6 element is 2,239 bp and only differs from dSpm-13 by a 2-bp deletion at the end of one of the 13-bp terminal inverted repeats. The effect of these deletions on the frequency and timing of Spm-induced excision is discussed herein. In the absence of Spm, each of the bz-m13 alleles conditions a nonmutant phenotype despite the presence of the insertions in the second exon. The role of RNA splicing in this phenomenon and the recent finding of an acceptor splice site within the terminal inverted repeat are also discussed.

Alleles↗
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↗
PubMed2003

A novel IS element, IS621, of the IS110/IS492 family transposes to a specific site in repetitive extragenic palindromic sequences in Escherichia coli.

An Escherichia coli strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. This insertion sequence (named IS621) has one large open reading frame encoding a putative protein that is 326 amino acids in length. A computer-aided homology search using the DNA sequence as the query revealed that IS621 was homologous to the piv genes, encoding pilin gene invertase (PIV). A homology search using the amino acid sequence of the putative protein encoded by IS621 as the query revealed that the protein also has partial homology to transposases encoded by the IS110/IS492 family elements, which were known to have partial homology to PIV. This indicates that IS621 belongs to the IS110/IS492 family but is most closely related to the piv genes. In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS621 belongs to the piv gene group, which is distinct from the IS110/IS492 family elements, which form several groups. PIV proteins and transposases encoded by the IS110/IS492 family elements, including IS621, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions. These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center. Interestingly, IS621 was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome. IS621 may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site. There are several elements belonging to the IS110/IS492 family that also transpose to specific sites in the repeated sequences, as does IS621. IS621 does not have terminal inverted repeats like most of the IS110/IS492 family elements. The terminal sequences of IS621 have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV. This suggests that IS621 initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites.

Amino Acid Sequence↗
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↗
PubMed2001

Micron, a microsatellite-targeting transposable element in the rice genome.

We have isolated a new family of mobile elements, Micron, which occur within microsatellites dispersed throughout the rice (Oryza sativa) genome. The first of these segments, Micron 001, was found in a microsatellite consisting of a (TA)n sequence upstream of the rice phytochrome A (phyA) gene. PCR analysis of related rice species suggests that Micron 001 integrated into this microsatellite locus prior to the divergence of the two wild species O. rufipogon and O. barthii from a common ancestor. Micron elements are short (393-bp), possess subterminal inverted repeats and the single strands have the potential to form stable secondary structures via several internal repeats. Aside from the absence of terminal inverted repeats, these characteristics resemble those of MITEs (Miniature Inverted-Repeat Transposable Elements). We estimate that 100-200 copies of Micron-related sequences are present in the rice nuclear genome, while the chloroplast and mitochondrial genomes lack this sequence. Nineteen homologs of Micron 001 exhibited extremely high nucleotide sequence conservation (greater than 90%), suggesting a recent spread of Micron elements within the genus Oryza. Surprisingly, nucleotide sequence alignments showed that all of the Micron elements are flanked on both sides by microsatellite sequence consisting mainly of (TA)n. Twenty-three elements were mapped to seven separate chromosomes. Therefore Micron elements form a family of dispersed, highly conserved repeats. This is the first report of a transposable element that targets microsatellite loci.

Amino Acid Sequence↗
PubMed1997

Conserved features of TBE1 transposons in ciliated protozoa.

The complete sequences of four TBE1 transposons from Oxytricha fallax and O. trifallax are presented and analyzed. Although two TBE1s are 98% identical to each other at the nucleotide level, the remaining two TBE1s are only 90% identical both to each other and to the other two. This large evolutionary divergence allows us to identify conserved TBE1 features. TBE1 transposons are 4.1 kbp long and are flanked by 3 bp target-site repeats. The elements consist of 78 bp inverted terminal repeats, of which the 17 terminal base pairs are Oxytricha telomere repeats; a central conserved section of 550 bp that includes a set of nested direct and inverted sequence repeats; and 3 open reading frames conserved for encoded amino acid sequence. The three open reading frames encode a 22 kDa basic protein of unknown function, a 42 kDa 'D,D35E' transposase, and a 57 kDa chimeric C2H2 zinc finger/protein kinase. The protein kinase domain of the 57 kDa protein is unusual, lacking a conserved ATP-binding motif.

Amino Acid Sequence↗
PubMed1996

Prt1, an unusual retrotransposon-like sequence in the fungus Phycomyces blakesleeanus.

This work reports the isolation and structural characterization of Prt1, a 4.7 kb retrotransposon-like sequence from the filamentous fungus Phycomyces blakesleeanus. Two open reading frames are found within Prt1. The first shows no similarity with known genes. The second encodes peptide stretches similar to the reverse transcriptase and RNaseH domains of the Ty3/gypsy family of LTR-retrotransposons. Prt1 lacks long terminal repeats, having instead short (54 bp) terminal inverted repeats. No target site duplication has been found. A single copy of Prt1 was detected in the genome of P. blakesleeanus. Adjacent to this sole copy of Prt1, a cluster of various short sequence repeats, both direct and inverted, is found. These sequences, which are reminiscent of defective, non-retroviral transposable elements, are also represented in other regions of the P. blakesleeanus genome.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100.

Insertion sequence IS100 was localized on a 9.5-kb plasmid of Yersinia pestis and was shown to be specific for Y. pestis and serotype I strains of Y. pseudotuberculosis. The nucleotide sequence of IS100 isolated from this plasmid was determined. The element, which was flanked by 5-bp direct repeats, contained 1953 bp including imperfect inverted terminal repeats of 52 and 61 bp long (43 bp were identical). Two open reading frames encoding potential polypeptides of 340 and 252 amino acids were identified on one DNA strand. Nucleotide sequence as well as deduced polypeptide sequences of IS100 were homologous to those of IS21, IS232 and IS640.

Base Sequence↗
PubMedDate not supplied

A homopolymer stretch composed of variable numbers of cytidine residues in the terminal repeats of infectious laryngotracheitis virus.

A homopolymer stretch composed of variable numbers of cytidine residues was found within the inverted terminal repeats of infectious laryngotracheitis virus (ILTV). A polymerase chain reaction procedure was developed to amplify a 750-bp fragment containing this homopolymer stretch. This fragment was then sequenced directly to determine the number of repeated cytidine residues in this homopolymer stretch, which could be used for strain differentiation. By this procedure, vaccine strains of tissue culture origin could be differentiated into two types: type I contains eight repeated cytidine residues, whereas type II contains 10 such residues. Vaccine strains of chicken embryo origin could also be divided into two types: type I contains mainly 11 repeated cytidine residues, whereas type II contains 15-21 such repeats. In comparison, two of the five field isolates examined contain 12-13 repeats; the other three field isolates contain 15-19 repeats, which were similar to the type II chicken-embryo-origin vaccines. The number of repeated cytidine residues described here could serve as a marker for the strain differentiation and epidemiologic study of ILTV.

Animals↗
PubMed1988

Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat.

The defective parvovirus, adeno-associated virus (AAV), contains a single-stranded DNA genome of 4681 bases with inverted terminal repeats of 145 bases. The distal 125 bases of the repeat are palindromic allowing a hairpin to form for initiation of DNA synthesis. The palindromic region contains three palindromes, two smaller internal palindromes flanked by a larger palindrome, which allow the hairpinned DNA to assume a T-shaped conformation during DNA replication. Deletion of an internal palindrome forming one of the crossarms of the T results in the inability of the AAV genome to be rescued from plasmid sequences and replicated. Restoration of the crossarm sequences with DNA that differs in primary sequence but maintains the symmetry of the palindrome results in viable AAV and propagation of the mutant sequences. In this paper we report further studies on the nature of mutants made within the crossarm of the T. Two types of substitution mutants were analyzed. Symmetrical sequence substitution mutants were viable as previously reported. An analysis of the kinetics of AAV DNA accumulation showed that the symmetrical sequence substitution mutants were indistinguishable from wild-type AAV. This was true if the AAV DNA was introduced into the cells either as plasmid DNA or as DNA extracted from virions. In contrast, intermolecular competition experiments showed either a dominance of the wild-type sequence or codominance of both sequences when both alleles were cotransfected into helper virus-infected cells. A preference for the wild-type sequence may also exist but is not required for efficient AAV replication. The second type of mutation studied was an asymmetrical sequence substitution mutant. This mutant was replicated but at a level too low to be propagated. These data suggest that symmetry is required in the internal palindromic region, presumably for the formation of the crossarm structure in the T-shape.

Cloning, Molecular↗