Molecular variation in Picea rubens and Picea mariana

Restriction fragment length polymorphism (RFLP) variation was examined among samples from the entire range of red spruce (Picea rubens Sarg.), the eastern complex of black spruce (Picea mariana (Mill.) B.S.P.), control-cross red-on-black hybrids, and natural populations of red and black spruce. Within-species and population variation was examined. In addition an accurate species index capable of identifying red spruce, black spruce and hybridization between the two species was developed. The nuclear rDNA repeat unit size in Picea ranged from a minimum of 32 kbp to greater than 40 kbp, two to three fold larger than the typical angiosperm rDNA unit. At a size greater than 32 kbp and a concentration averaging 1.2-1.3 $\times$ 10\sp4 copies per pg genomic DNA, the rDNA repeat constitutes approximately 4% of the spruce genome. The rDNA repeat units were found to be polymorphic within an individual genome with up to five distinct rDNA repeat unit types (alleles) evident. The RFLPs observed in the rDNA repeat were not species specific; however, noticeable trends in internal allelic frequencies were noticed which were useful for between-species differentiation. One marker (EMW 4.35) displayed a significant relationship with geographic origins and habitat suggesting that the observed between-species variation for this marker may be due to selection rather than the result of a true species difference. Variation in the nuclear rDNA repeat could not accurately differentiate hybrids from black spruce. Additional markers were required to identify hybrids. RFLPs were identified for the organelle genomes of red spruce, and black spruce. The organelle inheritance pattern was deduced using controlled-cross hybrids. Organelle markers were combined with allelic data from the nuclear rDNA repeat to derive a simple three character index capable of identifying red spruce, black spruce and hybridization between the two species. Significant gene flow was observed between red and black spruce populations located at Head Harbor, Isle au Haut, Maine but not on Mount Washington or Mount Lafayette. These findings suggest that hybridization and introgression between red and black spruce is influenced by not only proximity of the two species but also by habitat type

The Cycad Genotoxin MAM Modulates Brain Cellular Pathways Involved in Neurodegenerative Disease and Cancer in a DNA Damage-Linked Manner

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O6-methyldeoxyguanosine lesions, O6-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O6-mG DNA methyltransferase (MGMT) showed elevated O6-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer's disease

Five major nuclear ribosomal repeats represent a large and variable fraction of the genomic DNA of Picea rubens and P. mariana

The nuclear ribosomal repeats for the 18S, 5.8S, and 26S RNAs of two closely related Picea (spruce) species were characterized by restriction mapping and Southern blot hybridization. Restriction polymorphisms were identified in the IGS and ITS sequences; however, no polymorphism was species specific. As many as five different rDNA repeat units were observed in individual genomes. The repeat size for these gymnosperms ranged from a minimum of 32 kbp to greater than 40 kbp, two- to threefold larger than the typical angiosperm rDNA unit. Slot-blot hybridizations were used to determine the nuclear rDNA copy concentration. Among P. rubens individuals threefold variation was observed in the rDNA copy concentration, and among P. mariana individuals such variation was as much as sixfold. At a size greater than 32 kbp and at a concentration averaging 1.2-1.3 x 10(4) copies/pg, the rDNA constitutes approximately 4% of the total genome. Regression analysis revealed a significant relationship between copy concentration of the rDNA repeat unit in P. rubens and geographic origins. Differences in the rDNA content in Picea could contribute to the variation, in overall genome size, that has been observed within conifer species

Minimally Cytotoxic Doses of Temozolomide Produce Radiosensitization in Human Glioblastoma Cells Regardless of MGMT Expression

Decedent, a Texas resident, provided that if his wife elected to take under his will she would receive one-third of the total community property and one-third of his separate estate. The remaining two-thirds of decedent\u27s total estate was devised in trust for the benefit of his children. The widow elected to take under the will, thereby allowing her interest in the community property to pass as provided in the will. The executors claimed a marital deduction for the one-third separate property passing to the widow. Since she received less under the will than the value of her relinquished community property, the government disallowed the deduction. After paying the deficiency, the executors brought suit for a refund. The district court held that the bequest of one-third separate property qualified for the marital deduction. On appeal, held, affirmed, one judge dissenting. In determining the amount of the marital deduction, the value of the separate property passing to the surviving spouse is not reduced by the value of her relinquished community property passing under the will to decedent\u27s children. United States v. Stapf, 309 F.2d 592 (5th Cir. 1962), cert. granted, 372 U.S. 928 (1963)

Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma multiforme (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during radiotherapy and afterward has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O6-methylguanine (O6-meG) mediates GBM cell killing. Moreover, low or absent expression of O6-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O6-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O6-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O6-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O6-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA–glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets

Using nuclear and organelle DNA markers to discriminate among Picea rubens, Picea mariana, and their hybrids

The pattern of nuclear ribosomal alleles in controlled-cross black spruce × red spruce hybrids was compared with that of samples from provenance tests of the entire range of red spruce (Picearubens Sarg.) and the eastern complex of black spruce (Piceamariana (Mill.) BSP). In addition, restriction fragment length polymorphisms were identified for the organelle genomes of red spruce and black spruce. Organelle haplotypes were closely associated with species, but were not species specific. Data from controlled-cross hybrids suggest that mitochondria are maternally inherited and chloroplasts are paternally inherited in these spruce species. Organelle markers were combined with restriction fragment length polymorphism data from the nuclear rDNA repeat to derive a simple three-character index capable of identifying red spruce, black spruce, and hybrids of the two species

Hybridization between Picea rubens and Picea mariana: differences observed between montane and coastal island populations

Foliage was collected from natural stands of montane and island red spruce (Picearubens Sarg.) and black spruce (Piceamariana (Mill.) BSP) to examine within- and among-population genetic variation. Samples were scored for frequencies of nuclear ribosomal DNA (rDNA) alleles, and mitochondrial and chloroplast haplotypes. Samples were classified as red spruce, black spruce, or hybrid using two molecular methods: a three-character discriminant function based on molecular markers or a three-character molecular index. These results were found to be highly congruent with classification based upon a discriminant function using morphological traits. Among montane populations, hybridization and introgression between red and black spruce did not appear to be a major factor in the observed patterns of variation on elevational transects on Mount Washington and Mount Lafayette, N.H. However, extensive hybridization and introgression were detected among populations on Isle au Haut, Maine. The Mount Lafayette population displayed low variation in rDNA alleles compared with populations on Mount Washington and a range-wide provenance test in Stewartstown, N.H