ABSTRACT3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glycosylase not only for the cytotoxic 3MeA DNA lesion, but also for the mutagenic 1,N 6 -ethenoadenine (A) and hypoxanthine lesions. Aag appears to be the only 3MeA and hypoxanthine DNA glycosylase in liver, testes, kidney, and lung, and the only A DNA glycosylase in liver, testes, and kidney; another A DNA glycosylase may be expressed in lung. Although alkyladenine DNA glycosylase has the capacity to remove 8-oxoguanine DNA lesions, it does not appear to be the major glycosylase for 8-oxoguanine repair. Fibroblasts derived from Aag ؊͞؊ mice are alkylation sensitive, indicating that Aag ؊͞؊ mice may be similarly sensitive.In the face of inescapable DNA-damaging agents and inevitable spontaneous DNA degradation, the constant challenge to preserve genomic integrity has been met by the evolution of numerous pathways that protect against the genotoxic effects of DNA-damaging agents. Unless processed properly, DNA damage can be mutagenic, carcinogenic, and teratogenic, and DNA damage also may contribute to aging (1).Alkylating agents are found in our environment, in our food, inside all cells as natural metabolites, and in the clinic as cancer chemotherapeutic agents. Base excision repair (BER) is one of the major pathways for the repair of damaged DNA bases and proceeds through a sequence of reactions requiring several different enzymes. The first step involves excision of the damaged base (for which most cells are known to have several different DNA glycosylases). Base excision by glycosylases is followed by strand cleavage in the vicinity of the abasic site (by AP endonuclease or AP lyase), and preparation of the DNA ends for gap filling and ligation. DNA polymerase fills the gap, and DNA ligase seals the remaining nick, thus completing the BER process (1).Human and rat 3-methyladenine (3MeA) DNA glycosylases, so named because 3MeA was the first substrate identified for this class of enzymes (2), actually display an unexpectedly broad substrate range, including guanines methylated at the N3 or the N7 position (3-6), deaminated adenine [i.e., hypoxanthine (Hx)] (7), oxidized guanine 8-oxoguanine (8oxoG) (8), cyclic etheno adducts on both adenine and guanine (9, 10), and haloethylated purines (unpublished observations). The mouse alkyladenine DNA glycosylase (Aag) has not been assayed for release of all of these substrates, though it has been shown to act on N3 and N7 methylpurines and on 8oxoG (8,11). The precise biological effects of all of the DNA lesions repaired by mammalian 3MeA DNA glycosylases are not yet known for mammals, though there is strong evidence that 3MeA is cytotoxic (12), and other lesions may be mutagenic, namely Hx (13), 8oxoG (14), 1,N 6

The SNPWave marker system, based on SNPs between the reference accessions Colombia-0 and Landsberg erecta (Ler), was used to distinguish a set of 92 Arabidopsis accessions from various parts of the world. In addition, we used these markers to genotype three new recombinant inbred line populations for Arabidopsis, having Ler as a common parent that was crossed with the accessions Antwerp-1, Kashmir-2, and Kondara. The benefit of using multiple populations that contain many similar markers and the fact that all markers are linked to the physical map of Arabidopsis facilitates the quantitative comparison of maps. Flowering-time variation was analyzed in the three recombinant inbred line populations. Per population, four to eight quantitative trait loci (QTL) were detected. The comparison of the QTL positions related to the physical map allowed the estimate of 12 different QTL segregating for flowering time for which Ler has an allele different from one, two, or three of the other accessions.

In adult mammals numerous target tissues and organs for estrogens exist. Little is known about possible target organs during embryogenesis other than the reproductive tract and the gonads. This is the first report on the expression of estrogen receptor beta (ERbeta) in comparison with ERalpha mRNA during mouse embryogenesis. We found expression of estrogen receptor mRNA in the reproductive tract, but also in the atrial wall, brain, kidney, urethra, bladder neck, mammary gland primordium, midgut, cartilage primordia and perichondria.

We have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were significantly more sensitive to the toxic effects of the chemotherapeutic alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin and temozolomide than those from Mgmt wild-type mice. As expected, Mgmt-deficient fibroblasts and bone marrow cells were not sensitive to UV light or to the crosslinking agent mitomycin C. In addition, the 50% lethal doses for Mgmt -/- mice were 2- to 10-fold lower than those for Mgmt +/+ mice for 1,3-bis(2chloroethyl)-1-nitrosourea, N-methyl-N-nitrosourea and streptozotocin; similar 50% lethal doses were observed for mitomycin C. Necropsies of both wild-type and Mgmt -/mice following drug treatment revealed histological evidence of significant ablation of hematopoietic tissues, but such ablation occurred at much lower doses for the Mgmt -/- mice. These results demonstrate the critical importance of O6-methylguanine DNA methyltransferase in protecting cells and animals against the toxic effects of alkylating agents used for cancer chemotherapy.

Adverse trends in the reproductive health of male fish, including testis abnormalities and intersex gonads, have been increasingly reported over recent years. These effects have been associated with the exposure of fish to natural, synthetic, and xenobiotic estrogens present in the aquatic environment. A novel in vivo test system using transgenic zebrafish has been developed to rapidly determine the effects of estrogenic chemicals on critical life stages and sensitive target organs in the fish. In the transgenic zebrafish, an estrogen binding sequence linked to a TATA box and luciferase reporter gene was stably introduced. Binding of a substance to endogenous estrogen receptors (ER) and the subsequent transactivation of the ER result in luciferase gene induction that is easily measured in tissue lysates. Exposure to estradiol (E2) during juvenile stages of the transgenic zebrafish revealed the period of gonad differentiation to be the most responsive early life stage. In adult male transgenic zebrafish, the testis was the most sensitive and responsive target tissue to estrogens. Partial sequences of zebrafish estrogen receptor subtypes R and were cloned for the first time and were found to be differentially expressed in developing fish and tissues of adult male zebrafish. The transgenic zebrafish assay is a promising new tool to rapidly determine the estrogenic potency of chemicals in vivo.