scholarly journals Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction.

1986 ◽  
Vol 6 (12) ◽  
pp. 4295-4304 ◽  
Author(s):  
D B Roth ◽  
J H Wilson
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Short Sequence ◽  
End Joining ◽  
Nonhomologous Recombination ◽  
Sequence Homologies ◽  
Linear Molecules

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction

1986 ◽  
Vol 6 (12) ◽  
pp. 4295-4304
Author(s):  
D B Roth ◽  
J H Wilson
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Short Sequence ◽  
End Joining ◽  
Nonhomologous Recombination ◽  
Sequence Homologies ◽  
Linear Molecules

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Mechanisms of nonhomologous recombination in mammalian cells

1985 ◽  
Vol 5 (10) ◽  
pp. 2599-2607
Author(s):  
D B Roth ◽  
T N Porter ◽  
J H Wilson
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Nucleotide Sequencing ◽  
End Joining ◽  
Gene Encoding ◽  
Repair Synthesis ◽  
Nonhomologous Recombination

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.

scholarly journals Mechanisms of nonhomologous recombination in mammalian cells.

1985 ◽  
Vol 5 (10) ◽  
pp. 2599-2607 ◽  
Author(s):  
D B Roth ◽  
T N Porter ◽  
J H Wilson
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Nucleotide Sequencing ◽  
End Joining ◽  
Gene Encoding ◽  
Repair Synthesis ◽  
Nonhomologous Recombination

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.

scholarly journals Homologous and nonhomologous recombination in monkey cells.

1983 ◽  
Vol 3 (6) ◽  
pp. 1040-1052 ◽  
Author(s):  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Helper Virus ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 Large T Antigen ◽  
Nonhomologous Recombination

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.

Homologous and nonhomologous recombination in monkey cells

1983 ◽  
Vol 3 (6) ◽  
pp. 1040-1052
Author(s):  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Helper Virus ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 Large T Antigen ◽  
Nonhomologous Recombination

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.

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.

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts

1981 ◽  
Vol 1 (10) ◽  
pp. 919-931
Author(s):  
C L Cepko ◽  
U Hansen ◽  
H Handa ◽  
P A Sharp
Keyword(s):  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Coding Regions ◽  
Cell Extracts ◽  
Initiation Of Translation

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

scholarly journals WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases.

1997 ◽  
Vol 17 (8) ◽  
pp. 4877-4882 ◽  
Author(s):  
V V Ogryzko ◽  
P Wong ◽  
B H Howard
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
In Vivo Studies ◽  
Nuclear Antigen ◽  
Phase Progression

The p21(WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:386-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369:574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G1-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expression can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the simian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells. Proliferating-cell nuclear antigen (PCNA) overexpression was neither necessary nor sufficient to antagonize WAF1 action. Expression of the N-terminal domain of WAF1, responsible for cyclin-dependent kinase (CDK) interaction, had the same effect as full-length WAF1, while the PCNA binding C terminus exhibited modest activity. We conclude that S-phase progression in mammalian cells is dependent on continuing cyclin and CDK activity and that WAF1 affects S phase primarily through cyclin- and CDK-dependent pathways.

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells

1983 ◽  
Vol 3 (12) ◽  
pp. 2203-2210
Author(s):  
J W Innis ◽  
W A Scott
Keyword(s):  
Chromatin Structure ◽  
Plasmid Dna ◽  
Base Pair ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
Dnase I ◽  
Viral Origin ◽  
African Green Monkey Kidney

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.

Sign in / Sign up

Export Citation Format

Share Document