Malcolm J. Lippert

High‐levels of transcription through a gene stimulate spontaneous mutation rate, a phenomenon termed transcription‐associated mutation (TAM). While transcriptional effects on specific mutation classes have been identified using forward mutation and frameshift‐reversion assays, little is yet known about transcription‐associated base substitutions in yeast. To address this issue, we developed a new base substitution reversion assay (the lys2‐TAG allele). We report a 22‐fold increase in overall reversion rate in the high‐ relative to the low‐transcription strain (from 2.1‐ to 47‐ × 10⁻⁹). While all detectable base substitution types increased in the high‐transcription strain, G→T and G→C transversions increased disproportionately by 58‐ and 52‐fold, respectively. To assess a potential role of DNA damage in the TAM events, we measured mutation rates and spectra in individual strains defective in the repair of specific DNA lesions or null for the error‐prone translesion DNA polymerase zeta (Pol zeta). Results exclude a role of 8‐oxoGuanine, general oxidative damage, or apurinic/apyrimidinic sites in the generation of TAM G→T and G→C transversions. In contrast, the TAM transversions at GC base pairs depend on Pol zeta for occurrence implicating DNA damage, other than oxidative lesions or AP sites, in the TAM mechanism. Results further indicate that transcription‐dependent G→T transversions in yeast differ mechanistically from equivalent events in E. coli reported by others. Given their occurrences in repair‐proficient cells, transcription‐associated G→T and G→C events represent a novel type of transcription‐associated mutagenesis in normal cells with potentially important implications for evolution and genetic disease. Mol. Mutagen. 2013.

High levels of transcription in Saccharomyces cerevisiae are associated with increased genetic instability, which has been linked to DNA damage. Here, we describe a pGAL-CAN1 forward mutation assay for studying transcription-associated mutagenesis (TAM) in yeast. In a wild-type background with no alterations in DNA repair capacity, ≈50% of forward mutations that arise in the CAN1 gene under high-transcription conditions are deletions of 2–5 bp. Furthermore, the deletions characteristic of TAM localize to discrete hotspots that coincide with 2–4 copies of a tandem repeat. Although the signature deletions of TAM are not affected by the loss of error-free or error-prone lesion bypass pathways, they are completely eliminated by deletion of the TOP1 gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed into the context of a frameshift reversion assay, which is sensitive enough to detect Top1-dependent deletions even in the absence of high transcription. We suggest that the accumulation of Top1 cleavage complexes is related to the level of transcription and that their removal leads to the signature deletions. Given the high degree of conservation between DNA metabolic processes, the links established here among transcription, Top1, and mutagenesis are likely to extend beyond the yeast system.

High levels of transcription are associated with increased mutation rates in Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation (TAM). To obtain insight into the mechanism of TAM, we obtained LYS2 forward mutation spectra under low- versus high-transcription conditions in which LYS2 was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10 promoter, respectively. Because of the large size of the LYS2 locus, forward mutations first were mapped to specific LYS2 subregions, and then those mutations that occurred within a defined 736-bp target region were sequenced. In the low-transcription strain base substitutions comprised the majority (64%) of mutations, whereas short insertion-deletion mutations predominated (56%) in the high-transcription strain. Most notably, deletions of 2 nucleotides (nt) comprised 21% of the mutations in the high-transcription strain, and these events occurred predominantly at 5′-(G/C)AAA-3′ sites. No −2 events were present in the low-transcription spectrum, thus identifying 2-nt deletions as a unique mutational signature for TAM.

Membrane transport is a fundamental concept that undergraduate students of cell biology understand better with laboratory experience. Formal teaching exercises commonly used to illustrate this concept are unbiological, qualitative, or intricate and time consuming to prepare. We have developed an exercise that uses uptake of radiolabeled nutrient analogues by attachment-dependent animal cells cultured on multiwell trays. This system can readily be manipulated within a typical 3-h laboratory period to yield reproducible, biologically relevant, quantitative data regarding key aspects of membrane transport. Each 24-well tray of cultures allows a group of two to four students to compare eight conditions in triplicate. If different groups of students test different conditions or different types of cells, data can be shared for an even broader experience. The exercise is also readily adaptable for open-ended student projects. Here we illustrate the exercise measuring uptake of the nonmetabolizable glucose analogue [³H]-2-deoxy-d-glucose. Students successfully tested the effects of competing sugars, putative inhibitors of the GLUT1 transporter, and changes in cell physiology that might be expected to affect glucose transport in epithelial cells and fibroblasts. In this exercise students find the nutritional and medical implications of glucose transport and its regulation intriguing. They also learn to handle radioisotopes and cultured cells.

We postulated that gene fusions sometimes occur in normal cells as a result of gene rearrangements as have been observed involving oncogene loci in tumours. To test this, we searched for fusion-gene transcripts in selected human T-lymphocyte large deletion mutations of the hypoxanthineguanine phosphoribosyltransferase (hprt) gene using the 3′ rapid amplification of cDNA ends (RACE) technique. Aberrant hprt–containing transcripts were observed in seven out of 19 mutants (-36%) indicating that a surprising number of these rearrangements code for processed mRNAs. RNA splicing and polyadenylation occurred downstream of the non-deleted hprt sequence in chimeric transcripts and the majority resulted from mutants with fusions of hprt into regions containing a repetitive element (Alu, LINE or microsatellite).

To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine‐guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (M_f), and glycophorin A (GPA) variant erythrocytes (V_f) of both allele‐loss (ø/N) and allele‐loss‐and‐duplication (N/N) phenotypes in umbilical cord blood. The mean hprt M_f (1.40 ± 1.11 × 10⁻⁶, N = 66) and GPA V_f (ø/N 4.0 ± 2.2 × 10⁻⁶, N = 114; N/N 2.7 ± 2.0 × 10⁻⁶, N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt M_f was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 ± 0.41 × 10⁻⁶, N = 45, P < 0.01; t test, P < 0.01, Mann‐Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt M_f (0.70 ± 0.49 × 10⁻⁶) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann‐Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated M_f revealed a significant decrease in the relative contribution of gross structural mutations to the overall M_f (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, x² test), suggesting that the higher M_f resulted from an elevated level of “point” mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt M_f or GPA V_f.