scholarly journals Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

1992 ◽  
Vol 12 (10) ◽  
pp. 4422-4432 ◽  
Author(s):  
M D Baker ◽  
L R Read
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Mutant Cell ◽  
Immunoglobulin M ◽  
Constant Region ◽  
Wild Type ◽  
Ectopic Recombination ◽  
The Third ◽  
Homologous Recombination Event ◽  
Immunoglobulin Mu

We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell.

Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line

1992 ◽  
Vol 12 (10) ◽  
pp. 4422-4432
Author(s):  
M D Baker ◽  
L R Read
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Mutant Cell ◽  
Immunoglobulin M ◽  
Constant Region ◽  
Wild Type ◽  
Ectopic Recombination ◽  
The Third ◽  
Homologous Recombination Event ◽  
Immunoglobulin Mu

We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell.

Effects of mutation position on frequency of marker rescue by homologous recombination

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

scholarly journals Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

1986 ◽  
Vol 6 (11) ◽  
pp. 3948-3953 ◽  
Author(s):  
P Bullock ◽  
J Miller ◽  
M Botchan
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Somatic Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Recombination Product ◽  
Homologous Recombination Event ◽  
Sequencing Studies ◽  
Purine Pyrimidine

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.

scholarly journals Effects of mutation position on frequency of marker rescue by homologous recombination.

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613 ◽  
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells

1986 ◽  
Vol 6 (11) ◽  
pp. 3948-3953
Author(s):  
P Bullock ◽  
J Miller ◽  
M Botchan
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Somatic Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Recombination Product ◽  
Homologous Recombination Event ◽  
Sequencing Studies ◽  
Purine Pyrimidine

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.

scholarly journals Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching

2008 ◽  
Vol 205 (7) ◽  
pp. 1567-1572 ◽  
Author(s):  
Ali A. Zarrin ◽  
Peter H. Goff ◽  
Kate Senger ◽  
Frederick W. Alt
Keyword(s):  
B Cells ◽  
Heavy Chain ◽  
Recombination Event ◽  
Class Switch Recombination ◽  
Constant Region ◽  
Wild Type ◽  
Class Switch ◽  
Specific Factors ◽  
Antibody Class

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.

scholarly journals Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch

1989 ◽  
Vol 9 (5) ◽  
pp. 1850-1856
Author(s):  
W Dunnick ◽  
M Wilson ◽  
J Stavnezer
Keyword(s):  
Heavy Chain ◽  
Recombination Event ◽  
Point Mutations ◽  
Immunoglobulin M ◽  
Constant Region ◽  
Switch Region ◽  
Nucleotide Substitutions ◽  
Region Gene ◽  
Constant Region Gene ◽  
Switch Regions

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.

scholarly journals Correction of a deletion mutant by gene targeting with an adenovirus vector.

1993 ◽  
Vol 13 (2) ◽  
pp. 918-927 ◽  
Author(s):  
Q Wang ◽  
M W Taylor
Keyword(s):  
Homologous Recombination ◽  
Recombination Event ◽  
Recombinant Adenovirus ◽  
Adenovirus Vector ◽  
Adenovirus Type ◽  
Promoter Sequences ◽  
Homologous Recombination Event ◽  
Aprt Gene ◽  
Type Gene ◽  
Adenovirus Type 5

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.

scholarly journals Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch.

1989 ◽  
Vol 9 (5) ◽  
pp. 1850-1856 ◽  
Author(s):  
W Dunnick ◽  
M Wilson ◽  
J Stavnezer
Keyword(s):  
Heavy Chain ◽  
Recombination Event ◽  
Point Mutations ◽  
Immunoglobulin M ◽  
Constant Region ◽  
Switch Region ◽  
Nucleotide Substitutions ◽  
Region Gene ◽  
Constant Region Gene ◽  
Switch Regions

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.

scholarly journals Requirements for ectopic homologous recombination in mammalian somatic cells.

1996 ◽  
Vol 16 (12) ◽  
pp. 7122-7132 ◽  
Author(s):  
M D Baker ◽  
L R Read ◽  
B G Beatty ◽  
P Ng
Keyword(s):  
Dna Sequences ◽  
Somatic Cells ◽  
Immunoglobulin M ◽  
Hybridoma Cells ◽  
Gene Deletions ◽  
Homologous Chromosomes ◽  
Ectopic Recombination ◽  
Immunoglobulin Mu ◽  
Scanning Mechanism ◽  
In Cis

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.

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