Abstract— Light‐activated psoralens can covalently modify DNA and are widely used to study nucleic acid secondary structure and mutagenesis. Sequence specificity can be added to the photoaddition reaction by attaching the psoralen to an oligonucleotide designed to recognize a double‐stranded DNA binding site through formation of a triple helix. We have previously used this strategy to study targeted psoralen modification of a triplex binding site within the bacterial supF gene carried in viral genomes. In the present work we report the targeting of psoralen photoadducts in vitro to a specific site in the genome of a transgenic mouse. Both 10 base and 16 base oligonucleotide‐psoralen conjugates were capable of sequence‐specific modification of genomic mouse DNA, while a truncated 8 base conjugate was not. Light activation was necessary, and a dose dependence was demonstrated for target site modification and mutagenesis. The 10 base conjugate rapidly found its target, with sequence‐specific binding occurring after just 10 min incubation in the presence of mouse DNA. The ability to target psoralen photoadducts within mammalian genomes may prove useful in the study of chromatin structure and DNA repair. Moreover, this work may lead to potential in vivo applications of targeted psoralen modification.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from several different lines of transgenic mice were analyzed. One mouse line, 1139, was identified in which the frequency of spontaneous mutations was unusually high (3.15 x 10(-4)), 20-fold higher than in other transgenic mice carrying a similar number of copies of the lambda transgene (approximately 100). Over 75% of the spontaneous mutations from 1139 mice were found to be deletions, whereas mostly point mutations were recovered from the other mice. In 1139 no significant variation among adult tissues has been detected. However, embryonic tissue yielded a 3- to 4-fold lower frequency of mutations, most of which were point mutations rather than deletions. The frequency of mutations at another locus, the hypoxanthine phosphoribosyl transferase gene, was not elevated in fibroblast lines established in culture from the 1139 mice. Overall, these results suggest that the deletion mutagenesis affecting the transgene sequences in 1139 mice is a locus-specific effect occurring during growth and development. The increased mutagenesis could not be explained by the degree of methylation of the transgene sequences, since hypermethylation was seen in both 1139 mice and other mice with a low frequency of shuttle vector mutations. The integrated lambda vector DNA in 1139 mice was mapped to a single site on chromosome 7, but no mechanism for the mutagenesis was suggested by this localization. It is proposed that the lambda DNA may have either integrated into an unstable genomic site or created a newly unstable locus in the process of integration.

Oligonucleotides can bind in the major groove of duplex DNA and form triple helices in a sequence-specific manner , , , ). Progress in elucidating the third strand binding code has raised the possibility of developing nucleic acids as sequence-specific reagents for research and possibly clinical applications. Oligonucleotide-mediated triplex formation has been shown to prevent transcription factor binding to promoter sites and to block mRNA synthesis in vitro and in vivo (, , , ). Instead of using triplex formation to transiently block gene expression, however, we reasoned that it would be advantageous to use triple helix formation to target mutations to specific sites in selected genes in order to produce permanent, heritable changes in gene function and expression (, , ). In this approach, mutations are targeted to a selected site by linking the triplex-forming oligonucleotide to a mutagen so that the sequence specificity of the triplex formation can be imparted to the action of the mutagen (Fig. 1). Open image in new window Fig. 1. Strategy for triple helix-targeted mutagenesis. The 10-base triple helix-forming oligonucleotide is shown positioned above its target site in the supF gene (bp 167–176). The oligonucleotide is conjugated to a mutagen, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen, which is targeted to intercalate between basepairs 166 and 167, as indicated by the arrow. Photoactivation of the psoralen generates adducts and thereby mutations at the targeted site.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector carrying the supF mutation reporter gene (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 x 10(-5)), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.01). These results may help to elucidate pathways of endogenous mutagenesis in vivo, and they illustrate potentially important tissue-specific differences in genetic instability. (C) 1996 Wiley-Liss, Inc.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector carrying the supF mutation reporter gene (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 x 10(-5)), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.01). These results may help to elucidate pathways of endogenous mutagenesis in vivo, and they illustrate potentially important tissue-specific differences in genetic instability.

Psoralens are used clinically in the treatment of several skin diseases, including psoriasis, vitiligo, and cutaneous T cell lymphoma. However, psoralen treatment has been associated with an increased risk of squamous cell carcinoma of the skin. To elucidate molecular events that may play a role in the psoralen-related carcinogenesis, we examined psoralen-induced mutagenesis in a mouse fibroblast cell line carrying a recoverable, chromosomally integrated lambda phage shuttle vector. Using the supF gene as a mutation reporter gene, we determined the spectrum of mutations induced by photoactivation of 8-methoxypsoralen and of 5-methylangelicin. Both psoralens generated predominately T:A to A:T and some T:A to G:C transversions. Most of the mutations occurred at either 5' TpA or 5' ApT sites, both of which are conducive to interstrand cross-link formation. However, 5-methylangelicin produces only monoadducts, whereas 8-methoxypsoralen generated 20% cross-links and 80% monoadducts under the conditions of our experiments, as measured by direct HPLC analysis of the DNA from the treated cells. Although most of the mutations occurred at potentially cross-linkable sites, these results implicate monoadducts, as well as cross-links, as critical premutagenic lesions in psoralen-treated mammalian cells. These findings may help in the identification of carcinogenic changes induced by psoralen, and they may aid in the improved design of psoralen-based treatment regimens in the future.

A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167 - 176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.

Journal Article High efficiency, restriction-deficient in vitro packaging extracts for bacteriophage lambda DNA using a new E.coli lysogen Get access Edward J. Gunther, Edward J. Gunther Search for other works by this author on: Oxford Academic PubMed Google Scholar Noreen E. Murray, Noreen E. Murray 1Institute of Cell and Molecular Biology, The University of EdinburghDarwin Building, Mayfield Road, Edinburgh EH9 3JR, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Peter M. Glazer Peter M. Glazer * * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 21, Issue 16, 11 August 1993, Pages 3903–3904, https://doi.org/10.1093/nar/21.16.3903 Published: 11 August 1993 Article history Received: 23 June 1993 Accepted: 04 July 1993 Published: 11 August 1993

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering.