A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia cohl) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G C-A -T transitions; the other transition (A * T-G C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis oi UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.Recent studies on viral and cellular oncogenes have provided strong evidence that mutations can play a fundamental role in cellular transformation and carcinogenesis (3). In addition, many heritable diseases and developmental anomalies have a mutational origin. Although numerous mutagenic agents are known to interact with DNA, the molecular mechanisms by which these agents may cause mutations in mammalian cells are not well understood. To approach this problem, we have used a UV-irradiated simian virus 40 (SV40)-based shuttle vector to analyze the sequence specificities of point mutations induced in mammalian cells by DNA damage. We reasoned that a detailed knowledge of the characteristics and spectrum of the mutations formed would provide information about the molecular mechanisms of mutation induction.In our previous work on the replication of UV-damaged SV40 DNA in mammalian cells (1,32,34), we demonstrated that replication of damaged templates appears to occur by a dimer-bypass mechanism in which replication forks proceed beyond UV-induced lesions, leaving gaps (single-strand discontinuities of 50 to 150 bases) in the daughter strands. Replication is completed at the normal terminus, and relaxed circular gapped molecules are produced. These gapped molecules are then slowly converted to SV40 form I DNA. We proposed that these single-strand gaps would be filled by DNA synthesis which might include insertion of mismatched bases at the sites of UV-induced lesions in the template strands, leading to the induction of point mutations at the damage sites. An alternat...

In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis. This role can be separated genetically into three steps: (a) derepression of the SOS regulon by mediating the posttranslational cleavage of the LexA re-

Activity-dependent neuroprotective protein (ADNP, ∼123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using α-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by ∼1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/ neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

Previous reports identified two peptides that mimic the action of neuroprotective proteins derived from astrocytes. These peptides, NAPVSIPQ and SALLRSIPA, prevent neuronal cell death produced by electrical blockade, N-methyl-D-aspartate, and ␤-amyloid peptide (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). In the present study, all Damino acid peptides of NAPVSIPQ and SALLRSIPA were synthesized and compared respectively to the corresponding all L-amino acid peptides. In rat cerebral cortical test cultures cotreated with 1 M tetrodotoxin, the D-amino acid peptides produced similar potency and efficacy for neuroprotection as that observed for their respective L-amino acid peptides. Since all these peptides tested individually exhibited attenuation of efficacy at concentrations of Ͼ10 pM, combinations of these peptides were tested for possible synergies. Equimolar D-NAPVSIPQ and D-SALLRSIPA combination treatment produced potent neuroprotection (EC 50 , 0.03 fM) that did not attenuate with increasing concentrations. Similarly, the combination of L-NAPVSIPQ and D-SALLRSIPA also had high potency (EC 50 , 0.07 fM) without attenuation of efficacy. Combined administration of peptides was tested in a model of fetal alcohol syndrome and in a model of learning impairment: apolipoprotein E knockout mice. Intraperitoneal administration of D-NAPV-SIPQ plus D-SALLRSIPA to pregnant mice (embryonic day 8) attenuated fetal demise after treatment with an acute high dose of alcohol. Furthermore, oral administration of D-NAPVSIPQ plus D-SALLRSIPA significantly increased fetal survival after maternal alcohol treatment. Apolipoprotein E knockout mice injected with D-NAPVSIPQ plus D-SALLRSIPA showed improved performance in the Morris water maze. These studies suggest therapeutic potential for the combined administration of neuroprotective peptides that can act through a mechanism independent of chiral recognition.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.