Targeting the inverted CCAAT Box-2 of the topoisomerase IIalpha gene: DNA sequence selective recognition by a polyamide-intercalator as a staggered dimer

The synthesis and DNA binding characteristics of a polyamide-intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIalpha promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5'-AAGCTA-3'). Formation of ligand-induced CD bands at approximately 330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6x10(6) M(-1)), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 microM. DNaseI footprinting, using the topoIIalpha promoter sequence, highlighted the specificity of the conjugate for the cognate site (5'-AAGCTA-3'). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIalpha. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIalpha by blocking the binding of NF-Y.     

Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene

A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed.     

Treatment-resistant schizophrenia and DAT and SERT polymorphisms

One fifth to one third of all patients diagnosed with schizophrenia are resistant to drug treatment, which makes it a major clinical challenge. Genetic studies have focused on the association between treatment resistant schizophrenia (TRS) and a number of candidate genes, including serotonin and dopamine system genes. We explored associations between carefully characterized TRS and DAT–VNTR, SERT-PR and SERT-in2 polymorphisms. There were 173 patients enrolled in the study that were clinically evaluated using Positive and Negative Syndrome Scale and Clinical Global Impressions Scales and divided into two groups based on treatment resistance (92 patients in TRS group). Patients with a combination of SERT-in2 ll and DAT 9/10, 9/11, 9/9 and 6/6 genotypes were more likely to have TRS, compared to those with 10/10 or 10/12 genotype (OR = 5.1; 95% CI = 1.6–16.8). In the group of patients with DAT 10/10 or 10/12 genotype, those who also shared SERT-in2 ls or ss genotype were more likely to have TRS, compared to ll genotype carriers (OR = 2.7; 95% CI = 1.0–7.0). The model in which interaction between SERT-in2 and DAT polymorphisms is linked to TRS can possibly explain contradictory previous results regarding role of DAT and SERT in TRS, but further research is needed.     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

Overexpressing AtCKX1 in Potato Plants grown In Vitro: The Effects on Cytokinin Composition and Tuberization

Journal of Plant Growth Regulation - Over the last years, cytokinin deficiency has been studied in a variety of plant species, using transgenic expression of cytokinin oxidase/dehydrogenase genes...