scholarly journals Integration of a vector containing a repetitive LINE-1 element in the human genome.

1994 ◽  
Vol 14 (10) ◽  
pp. 6689-6695 ◽  
Author(s):  
M Richard ◽  
A Belmaaza ◽  
N Gusew ◽  
J C Wallenburg ◽  
P Chartrand
Keyword(s):  
Homologous Recombination ◽  
Human Genome ◽  
Mammalian Cells ◽  
Gene Families ◽  
Repeated Sequences ◽  
Mammalian Genomes ◽  
L1 Element ◽  
Multicopy Genes ◽  
The Stability ◽  
Reciprocal Process

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.

Integration of a vector containing a repetitive LINE-1 element in the human genome

1994 ◽  
Vol 14 (10) ◽  
pp. 6689-6695
Author(s):  
M Richard ◽  
A Belmaaza ◽  
N Gusew ◽  
J C Wallenburg ◽  
P Chartrand
Keyword(s):  
Homologous Recombination ◽  
Human Genome ◽  
Mammalian Cells ◽  
Gene Families ◽  
Repeated Sequences ◽  
Mammalian Genomes ◽  
L1 Element ◽  
Multicopy Genes ◽  
The Stability ◽  
Reciprocal Process

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.

scholarly journals Long CTG Tracts from the Myotonic Dystrophy Gene Induce Deletions and Rearrangements during Recombination at the APRT Locus in CHO Cells

2003 ◽  
Vol 23 (9) ◽  
pp. 3152-3162 ◽  
Author(s):  
James L. Meservy ◽  
R. Geoffrey Sargent ◽  
Ravi R. Iyer ◽  
Fung Chan ◽  
Gregory J. McKenzie ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Myotonic Dystrophy ◽  
Mammalian Cells ◽  
Triplet Repeats ◽  
Autosomal Dominant Disorder ◽  
Ctg Repeats ◽  
Dna Structures ◽  
High Frequencies ◽  
Large Numbers ◽  
The Stability

ABSTRACT Expansion of CTG triplet repeats in the 3′ untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG)17, (CTG)98, and (CTG)183 repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG)98 and (CTG)183, 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG)98 and (CTG)183 cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG)17 repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.

Gene Targeting

1999 ◽  
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Practical Approach ◽  
Simple Method ◽  
Mouse Es Cells ◽  
Cell Clones ◽  
Previous Edition ◽  
Pros And Cons

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

scholarly journals The M26 Hotspot of Schizosaccharomyces pombe Stimulates Meiotic Ectopic Recombination and Chromosomal Rearrangements

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1191-1204 ◽  
Author(s):  
Jeffrey B Virgin ◽  
Jeffrey P Bailey
Keyword(s):  
Homologous Recombination ◽  
Schizosaccharomyces Pombe ◽  
Dna Sequences ◽  
Chromosomal Rearrangements ◽  
Low Frequency ◽  
Repeated Sequences ◽  
Crossing Over ◽  
Ectopic Recombination ◽  
Recombination Hotspots ◽  
Integration Sites

Abstract Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10–1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35–60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.

Gene replacement with one-sided homologous recombination

1992 ◽  
Vol 12 (1) ◽  
pp. 360-367
Author(s):  
N Berinstein ◽  
N Pennell ◽  
C A Ottaway ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Target Gene ◽  
Gene Replacement ◽  
Immunoglobulin Genes ◽  
Nonhomologous Recombination ◽  
Immunoglobulin Locus ◽  
Chromosomal Sequences ◽  
Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

scholarly journals Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089 ◽  
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

scholarly journals Amifostine Metabolite WR-1065 Disrupts Homologous Recombination in Mammalian Cells

2010 ◽  
Vol 173 (2) ◽  
pp. 175-183 ◽  
Author(s):  
Jaroslaw Dziegielewski ◽  
Wilfried Goetz ◽  
Jeffrey S. Murley ◽  
David J. Grdina ◽  
William F. Morgan ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells
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