scholarly journals Extrachromosomal and chromosomal gene conversion in mammalian cells.

1986 ◽  
Vol 6 (5) ◽  
pp. 1608-1614 ◽  
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Insertion Mutation ◽  
Chromosomal Gene ◽  
Sequence Organization ◽  
Reciprocal Recombination ◽  
Homologous Fragment ◽  
Double Recombination ◽  
Neomycin Resistance ◽  
Chromosomal Recombination

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.

Extrachromosomal and chromosomal gene conversion in mammalian cells

1986 ◽  
Vol 6 (5) ◽  
pp. 1608-1614
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Insertion Mutation ◽  
Chromosomal Gene ◽  
Sequence Organization ◽  
Reciprocal Recombination ◽  
Homologous Fragment ◽  
Double Recombination ◽  
Neomycin Resistance ◽  
Chromosomal Recombination

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.

scholarly journals Suppression of Intrachromosomal Gene Conversion in Mammalian Cells by Small Degrees of Sequence Divergence

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1559-1568 ◽  
Author(s):  
Tamas Lukacsovich ◽  
Alan S Waldman
Keyword(s):  
Herpes Simplex ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Sequence Divergence ◽  
Insertion Mutation ◽  
Single Nucleotide ◽  
Heteroduplex Dna ◽  
Simplex Virus ◽  
Hsv Thymidine Kinase ◽  
Low Rate

Abstract Pairs of closely linked defective herpes simplex virus (HSV) thymidine kinase (tk) gene sequences exhibiting various nucleotide heterologies were introduced into the genome of mouse Ltk– cells. Recombination events were recovered by selecting for the correction of a 16-bp insertion mutation in one of the tk sequences. We had previously shown that when two tk sequences shared a region of 232 bp of homology, interruption of the homology by two single nucleotide heterologies placed 19 bp apart reduced recombination nearly 20-fold. We now report that either one of the nucleotide heterologies alone reduces recombination only about 2.5-fold, indicating that the original pair of single nucleotide heterologies acted synergistically to inhibit recombination. We tested a variety of pairs of single nucleotide heterologies and determined that they reduced recombination from 7- to 175-fold. Substrates potentially leading to G-G or C-C mispairs in presumptive heteroduplex DNA (hDNA) intermediates displayed a particularly low rate of recombination. Additional experiments suggested that increased sequence divergence causes a shortening of gene conversion tracts. Collectively, our results suggest that suppression of recombination between diverged sequences is mediated via processing of a mispaired hDNA intermediate.

scholarly journals Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology.

1989 ◽  
Vol 9 (4) ◽  
pp. 1621-1627 ◽  
Author(s):  
J Ellis ◽  
A Bernstein
Keyword(s):  
Gene Conversion ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Retroviral Vectors ◽  
Deletion Mutants ◽  
Gene Correction ◽  
Cell Clones ◽  
Infected Cells ◽  
Chromosomal Sequences ◽  
Neomycin Resistance

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology

1989 ◽  
Vol 9 (4) ◽  
pp. 1621-1627
Author(s):  
J Ellis ◽  
A Bernstein
Keyword(s):  
Gene Conversion ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Retroviral Vectors ◽  
Deletion Mutants ◽  
Gene Correction ◽  
Cell Clones ◽  
Infected Cells ◽  
Chromosomal Sequences ◽  
Neomycin Resistance

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

scholarly journals Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

2010 ◽  
Vol 30 (8) ◽  
pp. 1887-1897 ◽  
Author(s):  
Jeannine R. LaRocque ◽  
Maria Jasin
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Sequence Divergence ◽  
Dna Lesions ◽  
Human Cells ◽  
Deficient Mouse ◽  
Wild Type ◽  
Strand Breaks ◽  
Major Mechanism ◽  
Homeologous Recombination

ABSTRACT Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged (“homeologous”) sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally ≤100 bp, even in Msh2 − / − cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.

scholarly journals INDEPENDENT STRATUM FORMATION ON THE AVIAN SEX CHROMOSOMES REVEALS INTER-CHROMOSOMAL GENE CONVERSION AND PREDOMINANCE OF PURIFYING SELECTION ON THE W CHROMOSOME

Evolution ◽  
2014 ◽  
Vol 68 (11) ◽  
pp. 3281-3295 ◽  
Author(s):  
Alison E. Wright ◽  
Peter W. Harrison ◽  
Stephen H. Montgomery ◽  
Marie A. Pointer ◽  
Judith E. Mank
Keyword(s):  
Gene Conversion ◽  
Sex Chromosomes ◽  
Purifying Selection ◽  
Chromosomal Gene ◽  
W Chromosome

scholarly journals The search for homology does not limit the rate of extrachromosomal homologous recombination in mammalian cells.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 597-605
Author(s):  
A S Waldman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Insertion Mutant ◽  
Insertion Mutation ◽  
Homologous Pairing ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Homology Searching ◽  
Rate Limiting

Abstract Mouse LTK- cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini.

scholarly journals Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4509-4517
Author(s):  
P Hasty ◽  
J Rivera-Pérez ◽  
C Chang ◽  
A Bradley
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Homologous Sequence ◽  
Reciprocal Recombination ◽  
Replacement Vector ◽  
Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

Sign in / Sign up

Export Citation Format

Share Document