Carrie R. Valentine

Predicted changes in pre-mRNA secondary structure vary in their association with exon skipping for mutations in exons 2, 4, and 8 of the Hprt gene and exon 51 of the fibrillin gene.

Exon skipping that accompanies exonic mutation might be caused by an effect of the mutation on pre-mRNA secondary structure. Previous attempts to associate predicted secondary structure of pre-mRNA with exon skipping have been hindered by either a small number of available mutations, sub-optimal structures, or weak effects on exon skipping. This report identifies more extensive sets of mutations from the human and hamster Hprt gene whose association with exon skipping is clear. Optimal secondary structures of the wild-type and mutant pre-mRNA surrounding each exon were predicted by energy minimization and were compared by energy dot plots. A significant association was found between the occurrence of exon skipping and the disruption of a stem containing the acceptor site consensus sequences of exon 8 of the human Hprt gene. However, no change in secondary structure was associated with skipping of exon 4 of the hamster Hprt gene. Using updated energy parameters we found a different structure than that previously reported for exon 2 of the hamster Hprt gene. In contrast to the previously reported structure, no significant association was found between predicted structural changes and skipping of exon 2. For all three Hprt exons studied, there was a significantly greater number of deoxythymidine substitutions among mutations accompanied by exon skipping than among mutations without exon skipping. For exon 8, deoxythymidine substitution was also associated with structural changes in the stem containing the acceptor site consensus sequences. For exon 51 of the human fibrillin gene, structural differences from wild type were predicted for all four mutations accompanied by exon skipping that were not were predicted for a single mutation without exon skipping. Our results suggest that both primary and secondary pre-mRNA structure contribute to definition of Hprt exons, which may involve exonic splicing enhancers.

In vivo mutation analysis using the ΦX174 transgenic mouse and comparisons with other transgenes and endogenous genes

The ΦX174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo using single-burst analysis: determining the number of mutant progeny virus from an electroporated cell by dividing the culture into aliquots before scoring mutants. We review results obtained from single-burst analysis, including testing the hypothesis that high mutant frequencies (MFs) of G:C to A:T mutation recovered by transgenic targets include significant numbers of unrepaired G:T mismatches. Comparison between the ΦX174 and lacI transgenes in mouse spleen indicates that the spontaneous bps mutation frequency per nucleotide (mf(n)) is not significantly lower for ΦX174 than for lacI; the response to ENU is also comparable. For the lacI transgene, the spontaneous bps mf(n) is highly age-dependent up to 12 weeks of age and the linear trend extrapolates at conception to a frequency close to the human bps mf(n) per generation of 1.7 × 10(-8). Unexpectedly, we found that the lacI somatic (spleen) bps mf(n) per cell division at early ages was estimated to be the same as for the human germ-line. The bps mf(n) in bone marrow for the gpt transgene is comparable to spleen for the lacI and ΦX174 transgenes. We conclude that the G:C to A:T transition is characteristic of spontaneous in vivo mutation and that the MFs measured in these transgenes at early ages reflect the expected accumulation of in vivo mutation typical of endogenous mammalian mutation rates. However, spontaneous and induced mf(n)s per nucleotide for the cII gene in spleen are 5-10 times higher than for these other transgenes.

Frequency and spectrum of ENU-induced mutation in the ΦX174 transgene in mouse splenic lymphocytes and their significance to spontaneous transgenic rodent mutation frequencies

A perceived disadvantage of transgenic rodent mutation assays is that spontaneous mutant frequencies are high compared to those of endogenous genes and may consequently reduce sensitivity to induced mutation. We have previously argued that unrepaired G:T mismatches from spontaneous deamination of 5-methylcytosine at CpG sites could be converted to apparent in vivo mutations in the bacterial recovery systems because of rapid, random, mismatch repair in Escherichia coli. In this study, we have measured mutation frequencies in spleen of male mice induced by N-ethyl-N-nitrosourea (ENU) using the PhiX174 transgene, which is not subject to mismatch repair in E.coli, using single-burst analysis, a unique method to identify in vivo mutation. In order to compare our results to those using the lacI and cII transgenes, we converted all mutant frequencies to base pair substitution (bps) mutation frequencies per nucleotide based on mutant spectra from this study and published literature. We found this frequency in control spleen to be similar for lacI (3.8 +/- 0.7 x 10(-8)) and PhiX174 (3.1 +/- 1.2 x 10(-8)) at 6 weeks of age. We found a strong age dependence for spontaneous lacI mutation that extrapolated to a value at conception (1.8 +/- 0.9 x 10(-8)) that was not significantly different from the human germ line bps mutation frequency per nucleotide of 1.7 +/- 0.2 x 10(-8). These two transgenes provided similar mutational responses to 40 mg/kg ENU, 7- to 9-fold. In contrast, the cII target gene in the same tissue produces both spontaneous and induced mutation frequencies approximately 10 times higher, for unknown reasons. We conclude that the spontaneous mutant frequencies measured by the lacI and PhiX174 transgenes in this moderately dividing tissue accurately measure in vivo mutation frequencies at early ages. For these two transgenes, seemingly high mutant frequencies may reflect the expected accumulation of somatic mutation with age.