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.

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.

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.

The minimum amount of homology required for homologous recombination in mammalian cells

1984 ◽  
Vol 4 (11) ◽  
pp. 2253-2258
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Minimum Amount ◽  
Base Pairs ◽  
Viral Plaque

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

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.

scholarly journals Simian Virus 40 Large T Antigen Can Specifically Unwind the Central Palindrome at the Origin of DNA Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3312-3322 ◽  
Author(s):  
Weiping Wang ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Central Origin ◽  
Sv40 Dna

ABSTRACT The hydrophilic channels between helicase domains of simian virus 40 (SV40) large T antigen play a critical role in DNA replication. Previous mutagenesis of residues in the channels identified one class of mutants (class A: D429A, N449S, and N515S) with normal DNA binding and ATPase and helicase activities but with a severely reduced ability to unwind origin DNA and to support SV40 DNA replication in vitro. Here, we further studied these mutants to gain insights into how T antigen unwinds the origin. We found that the mutants were compromised in melting the imperfect palindrome (EP) but normal in untwisting the AT-rich track. However, the mutants' defect in EP melting was not the major reason they failed to unwind the origin because supplying an EP region as a mismatched bubble, or deleting the EP region altogether, did not rescue their unwinding deficiency. These results suggested that specific separation of the central palindrome of the origin (site II) is an essential step in unwinding origin DNA by T antigen. In support of this, wild-type T antigen was able to specifically unwind a 31-bp DNA containing only site II in an ATPase-dependent reaction, whereas D429A and N515S failed to do so. By performing a systematic mutagenesis of 31-bp site II DNA, we identified discrete regions in each pentanucleotide necessary for normal origin unwinding. These data indicate that T antigen has a mechanism to specifically unwind the central palindrome. Various models are proposed to illustrate how T antigen could separate the central origin.

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.

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 The minimum amount of homology required for homologous recombination in mammalian cells.

1984 ◽  
Vol 4 (11) ◽  
pp. 2253-2258 ◽  
Author(s):  
J Rubnitz ◽  
S Subramani
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Minimum Amount ◽  
Base Pairs ◽  
Viral Plaque

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

scholarly journals CMX001 (1-O-Hexadecyloxypropyl-Cidofovir) Inhibits Polyomavirus JC Replication in Human Brain Progenitor-Derived Astrocytes

2011 ◽  
Vol 55 (5) ◽  
pp. 2129-2136 ◽  
Author(s):  
Rainer Gosert ◽  
Christine Hanssen Rinaldo ◽  
Marion Wernli ◽  
Eugene O. Major ◽  
Hans H. Hirsch
Keyword(s):  
Human Brain ◽  
Host Cell ◽  
Cell Metabolism ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Brdu Incorporation ◽  
Sv40 Large T Antigen ◽  
Polyomavirus Jc

ABSTRACTPolyomavirus JC (JCV) replication causes progressive multifocal leukoencephalopathy (PML), a frequently fatal brain disease in immunodeficient patients, yet antiviral drugs are lacking. We characterized the lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding JCV (Mad-4) replication in human brain progenitor-derived astrocytes (PDA) and the simian virus 40 (SV40) large-T-antigen-expressing COS-7 cells up to 7 days postinfection (dpi). We examined JCV loads by PCR, the infection rate by immunofluorescence, and host cell toxicity by WST-1 and BrdU incorporation assays. Supernatants from CMX001-treated PDA demonstrated a drug concentration-dependent decrease in JCV loads and infectivity. CMX001 had only a modest effect on host cell metabolism but reduced overall BrdU incorporation. In PDA at 7 dpi, the CMX001 50% effective concentration (EC50) was 5.55 nM, the 50% cytotoxic concentration (CC50) was 184.6 nM, and the 50% selectivity index (SI50) was 33.3. The EC90was 19.7 nM, the CC90was 5,054 nM, and the SI90was 256.1. In COS-7 cells, JCV replication was faster and the EC50and EC90were 18- and 37-fold higher than those in PDA, i.e., 0.1 μM and 0.74 μM (CC50, 0.67 μM; SI50, 6.7; CC90, 12.2 μM; SI90, 16.5) at 5 dpi. We conclude that CMX001 inhibits JCV replication at concentrationsin vitrothat can be attained by oral administration without significant side effects in clinical studies.

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