Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.

Shortle, David; Nathans, Daniel

doi:10.1073/pnas.75.5.2170

Cited by 249 publications

(97 citation statements)

References 25 publications

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“…Previous studies have established the approximate boundaries of the minimal core region but have not pinpointed its precise limits (2,10,11,13,23,28,30). Internal deletions, insertions, and base substitutions have focused on a 23-bp palindrome in the center of the core origin (5,15,26,27). These mutants apparently interfere with DNA replication by altering Tantigen binding.…”

mentioning

confidence: 99%

Domain structure of the simian virus 40 core origin of replication.

Deb¹,

DeLucia²,

Baur³

et al. 1986

Mol. Cell. Biol.

163

172

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The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.Simian virus 40 (SV40) encodes a replicon that is highly host specific. This host dependence suggests that viral DNA replication depends on direct interactions of host factors with viral proteins or DNA and that the viral replicon may resemble cellular replicons. The viral origin consists of multiple distinct elements. An essential core origin is sufficient for autonomous replication, but three flanking regulatory elements provide related functions that increase the efficiency of replication more than 20-fold (2, 7, 9-11, 13, 17, 21, 23, 30). These elements are T-antigen-binding region I, the 21-base pair (bp) repeats, and the 72-bp enhancers. Each ancillary region is a component of the early promoteroperator that overlaps the core origin, and each may play a role in the organization of the open region of SV40 chromatin (4,13,14,17,19,33). A single viral protein, T antigen, binds to the core origin of replication to initiate each round of DNA synthesis (8,18,23,31,37). Cellular DNA polymerase alpha is also required for replication (9). The recent development of cell-free systems for accurate initiation of viral replication opens the way for identification of additional cellular proteins that interact with the viral replicon (22,29,38).

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mentioning

confidence: 99%

Domain structure of the simian virus 40 core origin of replication.

Deb¹,

DeLucia²,

Baur³

et al. 1986

Mol. Cell. Biol.

163

172

View full text Add to dashboard Cite

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“…Bisulfite treatment results in the deamination of cytosine residues to uracil residues (38). When NaHSO3-mutagenized DNA is reiaired in vitro before transfection, CG --TA transitions are usually observed (67). In this work, the mutagenized DNA was not repaired in vitro but rather was transfected directly.…”

Section: Methodsmentioning

confidence: 86%

“…NaHSO3 mutagenesis. NaHSO3 mutagenesis was as described by Shortle and Nathans (67), except that the concentration of NaHSO3, ( Quantitative nuclease S1 analysis of cytoplasmic and nuclear RNAs. HeLa suspension cells were infected at an MOI of 10 (unless otherwise indicated in the figure legends) with AdS or AdS mutants as described (7).…”

Section: Methodsmentioning

confidence: 99%

Transcription control region within the protein-coding portion of adenovirus E1A genes.

et al. 1984

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A single-base deletion within the protein-coding region of the adenovirus type 5 early region lA (EIA) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an ElA protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress EIA mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of ElA which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated EIA protein does not produce the cis-acting defect in EIA transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 ElA. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for ElA transcription during the late phase of infection.In Escherichia coli, promoter mutations generally result from changes in bases which interact directly with RNA polymerase and occur within 40 bases of the transcription initiation site (62, 68). In contrast to E. coli promoters, the DNA sequences which regulate the transcription of eucaryotic protein-coding genes, which are transcribed by RNA polymerase II, are more complex. The most highly conserved sequence element of polymerase II promoters, the TATA box (11; M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978), centered about 27 bases upstream from the transcription start site, probably interacts directly with the polymerase. Deletion of this sequence usually leads to a reduction in the rate of transcription initiation by a factor of 5 to 10, and the residual transcripts usually have heterogeneous cap sites (18,25,32,34,49,56), which are sites of transcription initiation (15,36). Transcription initiation in vitro in most instances is completely dependent on the TATA sequence (16,40,59,72,78). However, in eucaryotic cells, mutations at much greater distances from the start site than are the TATA box or the -35 region of E. coli promoters can have profound effects on transcription initiation (5, 18, 21, 33-35, 49, 50, 53, 71). Several strong viral promoter regions contain sequences called enhancers, which can increase transcription from a variety of promoters by an unknown mechanism which is largely independent of their position relative to the transcription start site (4,17,21,22,44,45,52,75). Since the function of these more distant transcription control signals is largely independent of their distance from the transcription initiation site, it seems unlikely that they interact directly with RNA polymerase II (44,48,52).In most genes transcribed by RNA polymerase II which have been analyzed, DNA sequences required for transcription initiation lie upstream from th...

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“…However, by the late 1970's alternative, more refined approaches had begun to emerge. For example, Shortle and Nathans (1978) were able to target such random mutagenesis to short, specific regions of DNA within a viral chromosome. Around this time, the first reports of successful sitedirected mutagenesis were also published.…”

Section: Introductionmentioning

confidence: 99%