DNA adducts, mutant frequencies, and mutation spectra in various organs of lambda lacZ mice exposed to ethylating agents.

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.

Regulation of expression of the Lactobacillus pentosus xylAB operon.

The xylose cluster of Lactobacillus pentosus consists of five genes, two of which, xylAB, form an operon and code for the enzymes involved in the catabolism of xylose, while a third encodes a regulatory protein, XylR. By introduction of a multicopy plasmid carrying the xyl operator and by disruption of the chromosomal xylR gene, it was shown that L. pentosus xylR encodes a repressor. Constitutive expression of xylAB in the xylR mutant is repressed by glucose, indicating that glucose repression does not require XylR. The xylR mutant displayed a prolonged lag phase compared to wild-type bacteria when bacteria were shifted from glucose to xylose medium. Differences in the growth rate in xylose medium at different stages of growth are not correlated with differences in levels of xylAB transcription in L. pentosus wild-type or xylR mutant bacteria but are positively correlated in Lactobacillus casei with a plasmid containing xylAB. Glucose repression was further investigated with a ccpA mutant. An 875-bp internal fragment of the ccpA gene of L. pentosus was isolated by PCR and used to construct a ccpA knockout mutant. Transcription analysis of L. pentosus xylA showed that CcpA is involved in glucose repression. CcpA was also shown to be involved in glucose repression of the alpha-amylase promoter of Lactobacillus amylovorus by demonstrating that glucose repression of the chloramphenicol acetyltransferase gene under control of the alpha-amylase promoter is strongly reduced in the L. pentosus ccpA mutant strain.