Histochemical GUS analysis.
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We showed that transposon P(GUS.p53.259H), mapped to chromosome 3 and carrying a dominant mutation p53(259)H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts- in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53(259)H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P(GUS.p53.259H) transposon itself. The effect of the P(GUS.p53.259H) appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo(alpha)pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P(GUS.p53.259H) effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts- clones induced by P(GUS.p53.259H) was established.
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The number of American children participating in youth sports within and outside of school settings has increased dramatically in recent years, leading to an increased number of sports-related injuries that include both physical and psychological problems. In this interview, Temple University Sports Medicine Centers' Ted Quedenfeld emphasizes that prevention is of paramount concern among those involved in youth athletic participation, and that nurses need to play a key role in prevention.
Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of beta-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate/insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. (131)I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate (K(in)) of (131)I-P-GUS in 2-day-old mice was 0.21 microl/g.min and decreased with age. By 7 weeks of age, transport of (131)I-P-GUS was not significant. Capillary depletion revealed that 62% of the (131)I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of (131)I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner. In contrast, the K(in) of (131)I-NP-GUS (0.04 microl/g.min) was significantly lower than (131)I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither (131)I-P-GUS nor (131)I-NP-GUS crossed the BBB in adult mice. These results indicate that (131)I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice.
4-coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. Transgenic Arabidopsis plants containing the At4CL1 or At4CL2 promoter fused to the beta-glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation of At4CL and other phenylpropanoid-specific genes, we generated ethyl methyl sulfate mutagenized populations of At4CL1::GUS and At4CL2::GUS transgenic lines and screened approximately 16,000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs enhanced the GUS-silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in the At4CL1::GUS and At4CL2::GUS silenced lines. While silencing in the seedlings of the At4CL1::GUS lines was root specific in seedlings, it affected all organs in the At4CL2::GUS lines. Also, At4CL1::GUS transgene silencing was confined to the transgene but At4CL2::GUS silencing extended to the endogenous At4CL2 gene. Organ-specific silencing of the At4CL1::GUS transgene cannot be explained by current models in the literature.
Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.