Amplification of a plasmid bearing a mammalian replication initiation region in chromosomal and extrachromosomal contexts
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Abstract:
Amplified genes in cancer cells reside on extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). We used a plasmid bearing a mammalian replication initiation region to model gene amplification. Recombination junctions in the amplified region were comprehensively identified and sequenced. The junctions consisted of truncated direct repeats (type 1) or inverted repeats (type 2) with or without spacing. All of these junctions were frequently detected in HSRs, whereas there were few type 1 or a unique type 2 flanked by a short inverted repeat in DMs. The junction sequences suggested a model in which the inverted repeats were generated by sister chromatid fusion. We were consistently able to detect anaphase chromatin bridges connected by the plasmid repeat, which were severed in the middle during mitosis. De novo HSR generation was observed in live cells, and each HSR was lengthened more rapidly than expected from the classical breakage/fusion/bridge model. Importantly, we found massive DNA synthesis at the broken anaphase bridge during the G1 to S phase, which could explain the rapid lengthening of the HSR. This mechanism may not operate in acentric DMs, where most of the junctions are eliminated and only those junctions produced through stable intermediates remain.Keywords:
Extrachromosomal DNA
Inverted repeat
Direct repeat
Extrachromosomal DNA
Microspora
Repeated sequence
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Inverted repeat
Transposase
Insertion sequence
Direct repeat
Pseudomonas syringae
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MDg3 is a family of mobile dispersed genetic elements represented by 15 copies in the haploid genome of D. melanogaster and flanked, like other similar elements, by the regions of homology. In the present work, these regions of mdg3 have been sequenced. The existence of perfect direct repeats 268 base pairs long has been demonstrated. Inverted repeats are located on the gene distal side of them. It is possible to construct a perfect 8 b.p. palindrome or a slightly mismatched 18 b.p. palindrome. The inverted repeats are flanked by two short 5 b.p. direct repeats.
Inverted repeat
Direct repeat
Homology
Melanogaster
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IS5075 and IS4321 are closely related (93.1% identical) members of the IS1111 family that target a specific position in the 38-bp terminal inverted repeats of Tn21 family transposons and that are inserted in only one orientation. They are 1,327 bp long and have identical ends consisting of short inverted repeats of 12 bp with an additional 7 bp (TAATGAG) or 6 bp (AATGAG) to the left of the left inverted repeats and 3 bp (AGA) or 4 bp (AGAT) to the right of the right inverted repeat. Circular forms of IS5075 and IS4321 in which the inverted repeats are separated by abutting terminal sequences (AGATAATGAG) were detected. A similar circular product was found for the related ISPa11. Transposition of IS4321 into the 38-bp target site was detected, but a flanking duplication was not generated. The precisely reconstituted target site was also identified. Over 50 members of the IS1111 family were identified. They encode related transposases, have related inverted repeats, and include related bases that lie outside these inverted repeats. In some, the flanking bases number 5 or 6 on the left and 4 or 3 on the right. Specific target sites were found for several of these insertion sequence (IS) elements. IS1111 family members therefore differ from the majority of IS elements, which are characterized by terminal inverted repeats and a target site duplication, and from members of the related IS110 family, which do not have obvious inverted repeats near their termini.
Inverted repeat
Direct repeat
Insertion sequence
Transposase
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Inverted repeat
Direct repeat
Autonomously replicating sequence
Sequence (biology)
Cloning vector
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Inverted repeat
Direct repeat
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Extrachromosomal DNA
Inverted repeat
Direct repeat
Autonomously replicating sequence
Scaffold/matrix attachment region
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Genomes of all organisms, especially of higher eukaryotes, contain a significant proportion of repeated sequences. A special group of inverted repeats are closely spaced inverted repeats known as palindromes (perfect palindromes) and pseuodopalindromes (imperfect palindromes). A palindrome is composed of two identical inverted repeats which are not separated, whereas inverted repeats in pseudopalindromes are not identical and/or they are separated by one or more base pairs (bp). Palindromes and pseudopalindromes can form DNA secondary structures which can lead to genetic diseases in human. In this study the influence of pseudopalindromes, i.e. the distance between inverted repeats on its recombinogenicity was investigated. As experimental organism yeast Saccharomyces cerevisiae was used. The results of this study showed that the insertion of 4 bp spacer DNA in the center of a 126 bp long palindrome does not reduce its recombinogenicity, but the 10 bp spacer DNA completely abolishes recombinogenicity of 126 and 150 bp long palindromes.
Inverted repeat
Direct repeat
Palindromic sequence
Repeated sequence
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Inverted repeat
Direct repeat
Insertion sequence
Transposase
Homology
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Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).
Direct repeat
Inverted repeat
Repeated sequence
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