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PubMed1990

Two domains in the terminal inverted-repeat sequence of transposon Tn3.

Tn3 and related transposons have terminal inverted repeats (IR) of about 38 bp that are needed as sites for transposition. We made mini-Tn3 derivatives which had a wild-type IR of Tn3 at one end and either the divergent IR of the Tn3-related transposon, gamma delta or IS101, or a mutant IR of Tn3 at the other end. We then examined both in vivo transposition (cointegration between transposition donor and target molecules) of these mini-Tn3 elements and in vitro binding of Tn3-encoded transposase to their IRs. None of the elements with an IR of gamma delta or IS101 mediated cointegration efficiently. This was due to inefficient binding of transposase to these IR. Most mutant IR also interfered with cointegration, even though transposase bound to some mutant IR as efficiently as it did to wild type. This permitted the Tn3 IR sequence to be divided into two domains, named A and B, with respect to transposase binding. Domain B, at positions 13-38, was involved in transposase binding, whereas domain A, at positions 1-10, was not. The A domain may contain the sequence recognized by some other (e.g., host) factor(s) to precede the actual cointegration event.

Base Sequence↗
PubMed1991

Molecular analysis of the Bg-rbg transposable element system of Zea mays L.

The two components of the Bg-rbg transposable element system of maize have been cloned. The Bg element, isolated from the mutable allele wx-m32:: Bg is inserted in the intron of the Waxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp. Bg has 5 bp terminal inverted repeats, and generates upon insertion an 8 bp direct duplication of the target sequence. Both ends of the Bg element contain a 76 bp direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCGGC is here repeated several times in direct or inverse orientation. The rbg element was isolated from the mutable allele o2m(r) where it is located in the promoter region of the Opaque-2 (O2) gene. rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of the Bg element, and is also flanked by an 8 bp direct duplication at the target site. Like Bg, rbg carries the 76 bp direct repeats. Restriction enzyme analysis reveals that, compared to Bg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between the rbg element and the autonomous Bg element.

Alleles↗
PubMed1986

DNA sequence analysis of a 5.27-kb direct repeat occurring adjacent to the regions of S-episome homology in maize mitochondria.

The DNA sequence of the 5270-bp repeated DNA element from the mitochondrial genome of the fertile cytoplasm of maize has been determined. The repeat is a major site of recombination within the mitochondrial genome and sequences related to the R1(S1) and R2(S2) linear episomes reside immediately adjacent to the repeat. The terminal inverted repeats of the R1 and R2 homologous sequences form one of the two boundaries of the repeat. Frame-shift mutations have introduced 11 translation termination codons into the transcribed S2/R2 URFI gene. The repeated sequence, though recombinantly active, appears to serve no biological function.

Amino Acid Sequence↗
PubMed1985

Mobile elements bounded by C4A4 telomeric repeats in Oxytricha fallax.

A novel family of micronuclear elements termed telomere-bearing elements (TBEs) is described. All 1900 family members are eliminated during macronuclear development. We conclude that they are transposons, first because the members are moderately conserved in sequence and probably dispersed in the genome. Second, in two cases, sequence comparison of the termini and flanks of the element with the corresponding empty site indicate that elements cause 3 bp target duplications (AAT) upon insertion; the 3 bp are part of the 5 bp target sequence, AATGA. Lastly, both elements carry 77 or 78 bp inverted terminal repeats. The tip of each inverted terminal repeat is the 17 bp telomere-like sequence 5' C1A4C4A4C4. At least half of the elements have these 17 bp or an extremely similar sequence. One possible pathway for transposition into new micronuclear sites starts in the developing macronucleus with excision to create a free linear form to which telomeres are added, followed by a low frequency of movement to the micronucleus, and insertion into the germ-line micronuclear DNA.

Animals↗
PubMed1989

Mu transposable elements are structurally diverse and distributed throughout the genus Zea.

The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of the Mu family of transposable elements. All characterized Mu elements contain similar approximately 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20-100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to the Mu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously cloned Mu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termed Mu4 and Mu5, have an organization characteristic of transposable elements and possess approximately 200-bp Mu terminal inverted repeats that flank internal DNA, which is unrelated to other cloned Mu elements. Mu4 and Mu5 are both flanked by 9-bp direct repeats as has been observed for other Mu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions of Mu4 and Mu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally from Mu-similar termini. The distribution of these elements in maize lines and related species suggests that Mu elements are an ancient component of the maize genome. Moreover, the structure of the Mu termini and the fact that Mu termini are found flanking different internal sequences leads us to speculate that Mu termini once may have been capable of transposing as independent entities.

Base Sequence↗
PubMed1983

Structure and expression of mouse VL30 genes.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.

Animals↗
PubMed1985

Origin of adenovirus DNA replication. Role of the nuclear factor I binding site in vivo.

An assay is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and a previously described assay, which measures multiple rounds of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or Ad4 genome termini were cotransfected with homologous and heterologous helper virus, and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in Ad4 indicated that, although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular protein nuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that this region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat.

Adenoviridae↗
PubMed1990

A transcript identified by MuA of maize is associated with Mutator activity.

A Mu element, which we designated MuA, was cloned from a maize line with a Mutator background by its homology to the terminal inverted repeats of Mu1. Like other Mu elements, MuA has terminal inverted repeats of approximately 200 bp which are homologous to those of Mu1, but the internal region is different. MuA is unique in several aspects, being approximately 5.5 kb in size and the largest Mu element reported to date. It is flanked by 8 bp duplications instead of the 9 bp duplications found in most other Mu insertions. The internal sequences of MuA hybridize to restriction fragments that cosegregate with Mutator activity in maize lines showing 1:1 segregation for somatic mutability. The most interesting observation is that a transcript of approximately 3.5 kb identified by the internal sequences of MuA is both qualitatively and quantitatively associated with Mutator activity. This transcript is present in maize lines containing germinal Mutator activity but is undetectable in maize inbreds with no known Mutator activity. The amount of the transcript is decreased in lines that have lost germinal Mutator activity. Northern analysis of maize a1-Mum mutant lines that segregate 1:1 for mutability shows that a transcript of the same size is associated with somatic Mutator activity. These data suggest that the 3.5 kb transcript is produced by the autonomous element that confers both germinal and somatic Mutator activity. The possibility that MuA is an autonomous or regulator element of the Mu family is discussed.

Blotting, Northern↗
PubMed1991

Capture of flanking DNA by a P element in Drosophila melanogaster: creation of a transposable element.

A 6.1-kilobase insertion into the rudimentary (r) gene was cloned and partially sequenced. The insertion consists of a 703-base-pair (bp) P element next to a 5.4-kilobase single-copy sequence. The normal position of the single-copy sequence is near the tip of the X chromosome. Upon insertion into the r gene, this chimeric element generated an 8-bp target-site duplication, characteristic of P elements. At the non-P-element end of the insertion, the first 8 bp are identical to the first 8 bp of the inverted terminal repeats of the P element. Thus, this element has inverted terminal repeats of 8 bp. This large element can excise from the r gene under conditions of hybrid dysgenesis, which indicates that it behaves like a normal P element. These data support the conclusion that a normally stable single-copy sequence has now become unstable and duplicated within the genome.

Animals↗
PubMed1992

The Tc2 transposon of Caenorhabditis elegans has the structure of a self-regulated element.

We have analyzed the sequence of the Tc2 transposon of the nematode Caenorhabditis elegans. The Tc2 element is 2,074 bp in length and has perfect inverted terminal repeats of 24 bp. The structure of this element suggests that it may have the capacity to code for a transposase protein and/or for regulatory functions. Three large reading frames on one strand exhibit nonrandom codon usage and may represent exons. The first open coding region is preceded by a potential CAAT box, TATA box, and consensus heat shock sequence. In addition to its inverted terminal repeats, Tc2 has an unusual structural feature: subterminal degenerate direct repeats that are arranged in an irregular overlapping pattern. We have also examined the insertion sites of two Tc2 elements previously identified as the cause of restriction fragment length polymorphisms. Both insertions generated a target site duplication of 2 bp. One element had inserted inside the inverted terminal repeat of another transposon, splitting it into two unequal parts.

Animals↗
PubMed1988

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.

Amino Acid Sequence↗
PubMed1987

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

Adenoviruses, Human↗
PubMed1989

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences.

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.

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↗
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↗
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↗