Insertion of unique and repetitive DNA fragments into the aprt locus of hamster cells

Nalbantoglu, Joséphine; Miles, Carol; Meuth, Mark

doi:10.1016/0022-2836(88)90535-9

Cited by 29 publications

(11 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Whenever DNA breaks were induced, the set of mutants isolated have included large insertions (Ͼ100 bp) of sequences of unknown origin. Examination of a set of 120 spontaneous APRT mutations identified only one 285-bp insertion of foreign DNA (14). As with our transposition mutation, the insertion mutations at the APRT locus showed no terminal repeats or duplication of target sites as would have been expected for a transposable element.…”

Section: Discussionsupporting

confidence: 62%

Transposition without transposase: a spontaneous mutation in bacteria

Rappleye

Roth

1997

J Bacteriol

View full text Add to dashboard Cite

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.Most spontaneous mutations can be sorted into four categories: base substitution, base addition or removal, and chromosomal rearrangements (deletions, duplications, inversions or transpositions). Rearrangements can arise by recombination between repeated sequences or can be catalyzed by transposases which act on conserved sequences at element ends. At low frequency, deletions and duplications can form without recombination between extensive repeats or transposases. Known bacterial transposition mutations are generated only by the activities of selfish elements.We describe here a spontaneous transposition event in Salmonella typhimurium that appears to have occurred without involvement of a transposable element or transposase. The mutation described moves a contiguous block of sequence (1.9 kb) and inserts it at a new site with no loss or duplication of the target sequence. While the system for detecting this mutant involved use of the transposable elements Tn10d(Tc) and Mud-lac, the mutational event itself occurred in strains devoid of any known transposase. Furthermore, the novel sequence junctions are not associated with the ends of any transposable element. Thus, the event may constitute a new type of spontaneous mutation. Rare events of this type may be difficult to detect without a specific selection system such as that used here. Spontaneous transposition events may have contributed to the evolution of hybrid proteins and multicistronic operons. MATERIALS AND METHODSBacterial strains. All strains are derivatives of S. typhimurium LT2 ( Table 1). The Tn10d(Tc) element is a transposition-defective derivative of transposon Tn10 lacking the transposase gene and the internal ends of IS10 (24). The MudJ element is a transposition-defective derivative of phage Mu described by Castilho et al. (3). Insertions of these elements in the eut operon have been described previously (18,20).Media. Complex medium was nutrient broth (NB) (0.8%; Difco Laboratories) supplemented with NaCl (0.5%). Minimal medium was Vogel and Bonner E medium (5) with added glucose (0.2%) as a carbon and energy source. Minimal E medium lacking citrate (NCE) (1) supplemented with 0.2% lactose was used in selecting mutants able to grow on lactose. The chromogenic ␤-galactosidase substrate 5-bromo-4-chloro-3-indoyl-␤-D-galactopyranoside (X-Gal) was added (final concentration, 20 g/ml) to solid NCE lactose medium to help visualize Lac ϩ colonies. An...

show abstract

Section: Discussionsupporting

confidence: 62%

Transposition without transposase: a spontaneous mutation in bacteria

Rappleye

Roth

1997

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Short direct repeats distinct from S, pentamers may also increase the likelihood for c-myc/IgHa rearrangements. In this respect, the excess of short recombinogenic motifs in the 5' region of c-myc such as the tetrameric sequences GAGG (13,25), CCCT (26), CGGC (27), and the pentameric GC stretches CGCGG/CCGCC (refs. 28 and 29; Fig.…”

Section: Discussionmentioning

confidence: 99%

Detection of recombinations between c-myc and immunoglobulin switch alpha in murine plasma cell tumors and preneoplastic lesions by polymerase chain reaction.

Janz

Müller

Shaughnessy

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Virtually all murine plasmacytomas carry chromosomal translocations that activate c-myc. The predominant (=90%) c-myc-activating chromosomal translocation in pristane (2,6,10,14-tetramethylpentadecane)-induced plasmacytomas in BALB/c mice is a reciprocal translocation t(12;15) in which an immunoglobulin heavy-chain switch sequence is joined to the 5' region of c-myc. The most common switch region involved is S.. We developed a direct PCR method to screen for recombinations between c-myc and S.. The critical step in establishing the method was the cloning and sequencing of the 5' flank of Ca, a region with a reduced number of switch repeats that is much more favorable for designing specific PCR primers than the highly repetitive S. region. In applying this PCR method, we detected translocation-specific junction fragments in transplanted (10/16, 63%) and primary (5/15, 33%) plasmacytomas. Moreover, the sensitivity of a nested version of that technique allowed us to discern rare t(12;15)s in BALB/c mice in the preneoplastic stage of plasmacytomagenesis (8/20 mice, 40%) as early as 30 days after administration of pristane. We conclude that t(12;15) is the probable primary, if not initiating, oncogenic step in plasmacytomagenesis.Plasmacytomas (PCTs) induced in BALB/cAnPt mice by the intraperitoneal (i.p.) injection of pristane (2,6,10,14-tetramethylpentadecane) (1) carry reciprocal chromosomal translocations t(12;15), t(6;15), or t(15;16) that are associated with the activation of the c-myc protooncogene (2). In the t(12;15) which occurs in 90% ofthe tumors the near 5' flanking region, the first exon or part of the first intron of c-myc is disrupted and joined head to head with genes in the immunoglobulin heavy chain (IgH) gene complex (3-6). The most common immunoglobulin gene breakpoint is in either the a-chain switch (Sa) region (7) The goal of the present study was to take advantage of these common breaksites in Sa/S'-Ca and devise a PCR method for detecting illegitimate-i.e., nonhomologous-recombinations between Sa/5'-Ca and c-myc. We describe here a direct PCR technique that detects 63% (10/16) of all translocation breakpoints in transplanted PCTs that are known to involve the Sa/5'-Ca,, region. We have applied this methodology to detect similar translocations in primary tumors and in the very early preneoplastic stages of PCT development.

show abstract

“…Once linear DNA has been joined end to end, it can be maintained in the cell in a large extrachromosomal array (27). When mammalian cells integrate DNA, they do so mainly nonhomologously (30), probably at transient DSBs (22,23). Similarly, in transfection assays using linearized viral DNA, end joining of the fragments is a prominent outcome in the recovered viable progeny (reviewed by Roth and Wilson [31]).…”

mentioning

confidence: 99%

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Nicolás¹,

Young²

1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

show abstract

Insertion of unique and repetitive DNA fragments into the aprt locus of hamster cells

Cited by 29 publications

References 29 publications

Transposition without transposase: a spontaneous mutation in bacteria

Transposition without transposase: a spontaneous mutation in bacteria

Detection of recombinations between c-myc and immunoglobulin switch alpha in murine plasma cell tumors and preneoplastic lesions by polymerase chain reaction.

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Contact Info

Product

Resources

About