Potassium accumulation as dynamic modulator of neurohypophysial excitability.

Activity-dependent modulation of excitable responses from neurohypophysial axons and their secretory swellings has long been recognized as an important regulator of arginine vasopressin and oxytocin release during patterned stimulation. Various activity-dependent mechanisms, including action potential broadening, potassium accumulation, and autocrine or paracrine feedback, have been proposed as underlying mechanisms. However, the relevance of any specific mechanism on net excitability in the intact preparation, during different levels of overall activation, and during realistic stimulation with trains of action potentials has remained largely undetermined. Using high-speed optical recordings and potentiometric dyes, we have quantified the dynamics of global excitability under physiologically more realistic conditions, that is in the intact neurohypophysis during trains of stimuli at varying frequencies and levels of overall activity. Net excitability facilitated during stimulation at low frequencies or at low activity. During persistent high-intensity or high-frequency stimulation, net excitability became severely depressed. Depression of excitable responses was strongly affected by manipulations of extracellular potassium levels, including changes to resting [K(+)](out), increases of interstitial spaces with hypertonic solutions and inhibition of Na(+)/K(+) ATPase activity. Application of the GABA(A) receptor blocker bicuculline or manipulations of Ca(2+) influx showed little effect. Numerical simulation of K(+) accumulation on action potentials of individual axons reproduced optically recorded population responses, including the overall depression of action potential (AP) amplitudes, modest AP broadening and the prominent loss of hyperpolarizing undershoots. Hence, extracellular potassium accumulation dominates activity-dependent depression of neurohypophysial excitability under elevated stimulation conditions. The intricate dependence on the short-term stimulation history and its resulting feedback on neurohypophysial excitability renders [K(+)](out) accumulation a surprisingly complex mechanism for regulating axonal excitability and subsequent neuroendocrine release.

Dhh1 regulates the G1/S-checkpoint following DNA damage or BRCA1 expression in yeast.

BACKGROUND: Heterologous expression of the tumor suppressor BRCA1 in the yeast Saccharomyces cerevisiae is lethal. To identify potential new BRCA1-interacting gene targets, we characterized highly conserved ionizing radiation (IR) sensitive gene deletions that suppress BRCA1-induced lethality in yeast. MATERIALS AND METHODS: Previously, we exposed an isogenic collection of yeast strains individually deleted for 4746 nonessential genes to IR and identified 199 radiation sensitive deletion strains. A subset (n = 130) of these were screened for those that suppressed the G1 arrest and lethality observed following galactose-induced expression from a GAL::BRCA1 plasmid in wild type yeast. RESULTS: We found that deletions of two core components of the highly conserved CCR4-NOT transcription complex (CCR4 or DHH1) rescued BRCA1-induced G1 arrest and lethality in yeast. This was not because of down regulation of the GAL promoter since both deletion strains produce large amounts of BRCA1 that is rapidly degraded. In addition, heterologous expression of BRCA1 results in increased transcription of the DNA damage-inducible reporter construct DIN::LacZ. Reduced viability following IR and nitrogen starvation was observed among strains deleted for CCR4 or DHH1 because of a defect in G1 to S phase checkpoint transition. Lethality following nitrogen starvation and IR was partially rescued in dhh1Delta strains by expressing the human ortholog of DHH1 (DDX6) which has been identified as a breakpoint oncogene.T CONCLUSIONS: hese results suggest that BRCA1 may promote genomic stability in human cells by interacting with the highly conserved ortholog of DHH1 (DDX6) to properly activate G1/S checkpoint arrest following DNA damage.

Genes required for ionizing radiation resistance in yeast.

The ability of Saccharomyces cerevisiae to tolerate ionizing radiation damage requires many DNA-repair and checkpoint genes, most having human orthologs. A genome-wide screen of diploid mutants homozygous with respect to deletions of 3,670 nonessential genes revealed 107 new loci that influence gamma-ray sensitivity. Many affect replication, recombination and checkpoint functions. Nearly 90% were sensitive to other agents, and most new genes could be assigned to the following functional groups: chromatin remodeling, chromosome segregation, nuclear pore formation, transcription, Golgi/vacuolar activities, ubiquitin-mediated protein degradation, cytokinesis, mitochondrial activity and cell wall maintenance. Over 50% share homology with human genes, including 17 implicated in cancer, indicating that a large set of newly identified human genes may have related roles in the toleration of radiation damage.

SIR functions are required for the toleration of an unrepaired double-strand break in a dispensable yeast chromosome.

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.

Neutrophil-dependent and neutrophil-independent alterations in the nasal epithelium of ozone-exposed rats.

Ozone induces epithelial hyperplasia and mucous cell metaplasia (MCM) in nasal transitional epithelium (NTE) of rats. A transient neutrophil influx accompanies upregulation of mucin messenger RNA (mRNA) before the onset of MCM. The present study was designed to examine the role of neutrophils in ozone-induced epithelial changes in the NTE of rats. Fourteen hours before inhalation exposure, male F344/N rats were injected intraperitoneally with antirat neutrophil antiserum to deplete circulating neutrophils, or were injected with normal (control) serum. Rats were then exposed to 0 ppm (filtered air) or 0.5 ppm ozone (8 h/d) for 1 or 3 d. Maxilloturbinates lined with NTE were analyzed to determine the epithelial labeling index; numeric densities of neutrophils, total epithelial cells, and mucous secretory cells; amount of stored intraepithelial mucosubstances; and steady-state ratMUC-5AC (mucin) mRNA levels. At 2 h after 3 d of exposure, rats treated with antiserum had 90% fewer circulating neutrophils than did rats treated with control serum. Antiserum-treated, ozone-exposed rats had 87% fewer infiltrating neutrophils than did control serum-treated, ozone-exposed rats. At 4 d after 3 d of exposure, antiserum-treated, ozone-exposed rats had 66% less stored intraepithelial mucosubstances and 58% fewer mucous cells in their NTE than did control serum-treated, ozone-exposed rats. Antiserum treatment had no effects on ozone-induced epithelial cell proliferation or mucin mRNA upregulation. The results of this study indicated that ozone-induced MCM was neutrophil-dependent, whereas ozone-induced epithelial cell proliferation and mucin gene upregulation were neutrophil-independent.

Effects of pre-existing rhinitis on ozone-induced mucous cell metaplasia in rat nasal epithelium.

Ozone causes rhinitis and nasal epithelial alterations. The toxicity of ozone on nasal airways with pre-existing rhinitis has not been investigated. The present study was designed to determine the effect of endotoxin-induced rhinitis on ozone-induced epithelial alterations, especially mucous cell metaplasia (MCM), in the nasal transitional epithelium (NTE) of rats. Six h prior to daily inhalation exposure, male F344/N rats were intranasally instilled with saline or endotoxin (100 microgram/day). Rats were killed 2 h or 4 days after 3-day (8 h/day) exposure to ozone (0.5 ppm) or filtered air (0 ppm). The maxilloturbinate from one nasal passage was processed for morphometric analyses of the numbers of neutrophils and epithelial cells and the amount of intraepithelial mucosubstances (IM) in the NTE. The maxilloturbinate from the other nasal passage was processed for a mucin-specific (rMuc-5AC) mRNA analysis. At 2 h postexposure, endotoxin/ozone-exposed rats had 48 and 3 times more neutrophils in the NTE than did saline/air- and saline/ozone-exposed rats, respectively. Ozone-exposed rats had 35% more NTE cells and 2-fold more mucin mRNA than did saline/air-exposed rats, independent of endotoxin exposure. At 4 days postexposure, endotoxin/ozone-exposed rats had 5 and 2 times more IM and mucous cells, respectively, than did saline/air- and saline/ozone-exposed rats. Though endotoxin/air-exposed rats killed at 2 h postexposure had more neutrophils (40-fold), epithelial cells (27%) and mucin mRNA (2-fold) in the NTE than did saline/air-exposed rats, no MCM was present in those rats killed at 4 days postexposure. The results of the present study indicated that pre-existing rhinitis augments ozone-induced MCM.

Long-lasting effects of chronic ozone exposure on rat nasal epithelium.

Ozone, the principal oxidant pollutant in photochemical smog, causes airway epithelial injury in the upper and lower respiratory tract of laboratory animals. We have recently reported that long-term inhalation exposure to ozone causes mucous-cell metaplasia (MCM) in the surface epithelium lining the nasal airways of F344 rats. The principal objective of the present study was to determine the persistence of ozone-induced MCM in the nasal epithelium after the end of a chronic exposure. Male F344/N rats were exposed to 0, 0.25, or 0.5 ppm ozone, for 8 h/d, 7 d/wk for 13 wk. Animals were killed 8 h, 4 wk, or 13 wk after the end of the chronic exposure. Ozone-related alterations in the nasal epithelium were qualitatively and quantitatively characterized through histochemistry, image analysis, and morphometric techniques. Some rats were exposed for an additional 8 h to 0.5 ppm ozone at 13 wk after the end of the chronic exposure to determine whether previous ozone exposure results in persistent changes in the sensitivity of nasal epithelium to acute injury. At the end of the chronic exposure, hyperplasia was present in the nasal epithelium of rats exposed to 0.25 and 0.5 ppm ozone. By 13 wk postexposure, this proliferative alteration was still evident only in the rats exposed to 0.5 ppm ozone. Ozone-induced MCM with associated intraepithelial mucosubstances was evident only in the nasal tissues of rats exposed to 0.5 ppm ozone. Though attenuated, these alterations in the nasal mucous apparatus were still detectable at 13 wk after the end of the exposure. At this same time after the chronic exposure, an acute (8 h) exposure to 0.5 ppm ozone induced an additional increase of mucosubstances in the nasal epithelium of rats previously exposed to 0.5 ppm ozone, but not in rats chronically exposed to 0 or 2.5 ppm ozone. The persistent nature of the ozone-induced MCM in rats documented in this report suggests that ozone exposure may have the potential to induce similar long-lasting alterations in the airways of humans.

A persistent double-strand break destabilizes human DNA in yeast and can lead to G2 arrest and lethality.

Double-strand breaks (DSBs) are an important source of genomic change in many organisms. We have examined the consequences of a persistent versus a rapidly repaired DSB on cell progression, viability, and stability of human DNA contained in dispensable yeast artificial chromosomes (YACs) within the yeast Saccharomyces cerevisiae. An Alu-URA3-YZ integrating plasmid was used to target the YZ sequence to repetitive Alu sequences within the human YAC. The YZ site can be cut by an inducible HO-endonuclease resulting in a DSB. Two classes of DSBs had been identified previously: those that could be rapidly repaired (RR-DSB), through recombination between flanking Alus; and persistent DSBs (C. B. Bennett et al., Mol. Cell. Biol., 16: 4414-4425, 1996). These persistent DSBs (type 1) resulted in G2 delay and lethality. A third class of DSB is now identified corresponding to a persistent DSB that does not lead to G2 arrest or lethality (type 2). Unlike YACs in which the DSB was rapidly repaired, the two types of persistent DSBs destabilized the human YAC DNA, resulting in a high likelihood of YAC loss (approximately 85% of surviving colonies). Furthermore, both types of persistent DSBs could be misrepaired, resulting in mostly large internal or terminal deletions in the retained YACs. Therefore, recovery of these altered YACs can occur regardless of the effect of the DSBs on G2 arrest and cell lethality. If similar events occur in mammalian cells, persistent DSBs could be the initiating events that lead to a loss of heterozygosity and the expression of recessive oncogenes seen in malignant cells.

A double-strand break within a yeast artificial chromosome (YAC) containing human DNA can result in YAC loss, deletion or cell lethality.

Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12). An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site. The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G2 delay associated with the persistent DSBs. The presence of persistent DSBs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break. For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl. Acad. Sci. USA 90:5613-5617, 1993). However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study. The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.

Do caries explorers transmit infections with persons? An evaluation of second molar caries onsets.

UNLABELLED: Dental caries explorers may become contaminated during routine caries examinations with pathogenic organisms and thereby potentially transmit infections from one tooth to another within a patient. The purpose of this study was to test the hypothesis that the contamination status of explorers influenced the caries risk of second molars. Two explorer contamination statuses were defined: (1) contamination status 1--explorers which had probed a carious molar just prior to examining the second molar, and (2) contamination status 2--sterile explorers versus explorers which had probed several teeth. Caries examinations were performed by 4 dentists on a cohort of 4th grade students in Belize City. The examination dates and sample sizes (n) were: September-October 1989 (n = 1,277), January 1991 (n = 1,111), and January 1992 (n = 961), and January-February 1993 (n = 861). Within this cohort, there were 221 subjects who (1) had at least one pit and fissure carious onset on a caries-free second molar, (2) had no evidence of dental treatments, and (3) were examined by the same examiner during the entire study. After adjusting for confounding variables, the examination of a second molar with a dental caries explorer in either contamination status 1 or 2 had no substantial effect on the caries risk (rate ratio 0.95, 95% confidence interval: 0.77-1.18, and rate ratio 1.18, 95% confidence interval: 0.89-1.56, respectively). If a true rate ratio of 1.7 or greater was associated with the contamination status 1 and 2, these analyses had more than 99 and 80% probability of detecting it, respectively. CONCLUSIONS: Examining a sound second molar with a contaminated dental explorer either does not affect the caries risk, or results in such a small increase in caries risk that it can only be reliably identified in studies where the exposure of sound teeth to contaminated dental explorers is randomized.

Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid.

Cells of the yeast Saccharomyces cerevisiae are delayed in the G2 phase of the cell cycle following chromosomal DNA damage. This arrest is RAD9-dependent and suggests a signaling mechanism(s) between chromosomal lesions and cell cycling. We examined the global nature of growth inhibition caused by an HO endonuclease-induced double-strand break (DSB) at a 45-bp YZ sequence (from MAT YZ) in a non-yeast region of a dispensable single-copy plasmid. The presence of an unrepaired DSB results in cellular death even though the plasmid is dispensable. Loss of cell viability is partially dependent on the RAD9 gene product. Following induction of the DSB, 40% of RAD+ and 49% of rad9 delta cells [including both unbudded (G1) and budded (S plus G2) cells] did not progress further in the cell cycle. The remaining RAD+ cells progressed to form microcolonies (< 30 cells) containing aberrantly shaped inviable cells. For the rad9 delta mutant, the majority of the remaining cells produced viable colonies accounting for the greater survival of the rad9 delta strain. Based on the profound effects of a single nonchromosomal DNA lesion, this system provides a convenient means for studying the signaling effects of a DNA lesion, as well as for designing strategies for modulating cell proliferation.

Genetic requirements for frameshift reversion induced by bulky DNA adducts in M13 DNA.

In order to analyze the genetic requirements and mechanisms of frameshift mutagenesis by activated aflatoxin B1 (AFB1), in vitro-modified phage M13 replicative form (RF) DNA was transfected into appropriate Escherichia coli cells and +1 or -1 frameshift revertants in the lacZ(alpha) gene were isolated. This analysis shows that both +1 and -1 frameshift mutagenesis by AFB1 is significantly reduced in a umuC- background. On the other hand, in the absence of RecA, +1 frameshift mutagenesis is partially reduced, but -1 frameshift mutagenesis is unaffected. DNA sequence analysis of +1 frameshifts induced by AFB1 in recA- cells suggests that the mutations occur at the same sites as in recA+ cells, but that there are significant differences in the specificity of the observed base changes. A model consistent with the observed effects in the absence of RecA suggests that an appreciable fraction of AFB1-adducted guanines can correctly template for a cytosine.

DNA damage and biological expression of adenovirus: a comparison of liquid versus frozen conditions of exposure to gamma rays.

Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.

Effects of SOS and MucAB functions on reactivation and mutagenesis of M13 replicative form DNA bearing bulky lesions.

We have previously determined the specificity of -1 frameshifts induced by aflatoxin-B1-2,3-dichloride (AFB1C12) in phage M13 double-strand replicative form (RF) DNA. The system consists of: (i) in vitro adduction of RF DNA of BK8, a lacZ + 1 frameshift derivative of phage M13mp8; (ii) transfection into unirradiated or UV-irradiated bacterial host cells; (iii) scoring and sequencing of revertants (i.e., -1 frameshifts). The previous data had shown that induction of SOS functions enhanced mutagenesis. However, this increase in mutagenesis is not accompanied by enhanced survival in a majority of the strains tested. Here, we present evidence to show that the lack of SOS reactivation is a specific property of the RF DNA system rather than a specific property of the lesion. A model mechanism based on the replicative strategy of transfected RF DNA can account for these observations. In addition, we have calculated individual Weigle mutagenesis factors at 8 major mutagen induced sites reported previously. Analysis of these data indicates that, within a restricted subset of possible mutational events (i.e., -1 frameshifts), Weigle mutagenesis is affected by both the DNA sequence environment of the mutation site as well as the repair phenotype of the cell.

Delayed expression of enhanced reactivation and decreased mutagenesis of UV-irradiated adenovirus in UV-irradiated ataxia telangiectasia fibroblasts.

Fibroblasts from patients with ataxia telangiectasia (AT) are hypersensitive to the lethal effects but hyposensitive to the mutagenic effects of ionizing radiation, suggesting that AT cells may be defective in some process which modifies damage to DNA. In this study we have examined the UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis (UVEM) of UV-irradiated adenovirus in AT fibroblasts. UVER was examined using both V antigen expression as well as progeny production from infected cell cultures. Viral mutagenesis was studied by examining the induction of phenotypically wild-type revertants among the progeny obtained from fibroblasts infected with a temperature-sensitive early mutant of adenovirus (Ad5ts36). UVER factors for Ad V antigen expression were significantly less than normal in the AT strains tested when infection occurred immediately after UV-irradiation of cells. However, UVER factors were greater than 1 and similar to those found for normal strains when cells were infected 24 h after UV-irradiation, indicating a delay in the expression of UVER for Ad V antigen in AT cells. UV-irradiation of both normal and AT cells 24 h prior to infection also resulted in a significant increase in progeny survival for UV-irradiated Ad. In normal cells, this progeny UVER was concomitant with a significant increase in the mutation frequency for UV-irradiated virus (increase in targeted mutagenesis) suggesting the existence of an inducible error-prone DNA repair mode in normal human cells. In contrast, pre-UV-irradiation of AT cells resulted in a significant decrease in the mutation frequency for UV-irradiated virus. These results suggest that AT cells lack an inducible error-prone DNA mode and that the delayed expression of UVER in AT cells results from a relatively error-free mechanism.

Enhanced reactivation and mutagenesis of UV-irradiated adenovirus in normal human fibroblasts.

UV-enhanced reactivation (UVER) and UV-enhanced mutagenesis (UVEM) for two adenovirus temperature-sensitive mutants (Ad5ts35 and Ad5ts125) were examined following the infection of normal human fibroblasts. Fibroblast monolayers were either UV-irradiated or left non-irradiated and subsequently infected with either non-irradiated or UV-irradiated virus. After incubation of the infected cultures at the permissive temperature, the induction of wild-type revertants in the viral progeny was determined by plaquing at the permissive (33 degrees C) and the non-permissive (39 degrees C) temperatures on human HeLa or KB cells. UV-irradiation of the virus alone resulted in a dose-dependent increase in the UV-induced reversion frequency (RF) of viral progeny and a dose-dependent exponential decrease in progeny survival, when infecting non-irradiated cells. Analysis of the slopes of the UV-induced reversion curves suggested that 2.5 +/- 0.3 and 2.4 +/- 0.5 'hits' were required to produce a targeted reversion event among the viral progeny of Ad5ts36 and Ad5ts125 respectively. UV-irradiation of cells 24 h prior to infection resulted in a significant increase in progeny survival for UV-irradiated virus (UVER factor = 3.4 +/- 0.8) concomitant with a significant increase in RF for UV-irradiated virus (targeted increase = 1.9 +/- 0.3). The UV-induced RF per lethal hit to the virus was also significantly greater in UV-irradiated compared with non-irradiated cells. These results are consistent with the existence of a UV-inducible error-prone DNA repair mechanism in normal human cells.

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride.

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.