A method to select for mutator DNA polymerase deltas in Saccharomyces cerevisiae.

Proofreading DNA polymerases share common short peptide motifs that bind Mg(2+) in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase delta mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase delta mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.     

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

A protocol for genetic transformation mediated by Agrobacterium tumefaciens and production of transgenic Dendrobium lasianthera has been developed for the first time. The 8-week-old protocorm explants were used as target of transformation with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pG35SKNAT1. Several parameters such as infection period, Agrobacterium density, concentration of acetosyringone, and co-cultivation period were evaluated for the transformation efficiency. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) with p?<?0.05. Subsequently, KNAT1 gene expression was confirmed by polymerase chain reaction (PCR) analysis. The highest efficiency of transformation (70%) obtained from protocorm explants infected with Agrobacterium culture was at the OD₆₀₀ concentration of 0.6 for 30?min, and co-cultivated with acetosyringone 100?µM for 5?days. The results of confirmation by PCR analysis show that the KNAT1 gene has been integrated and expressed in the genome of Dendrobium lasianthera transgenic.     

The effect of cadmium on plasma melanocyte-stimulating hormone and morphological changes of melanophores in the cichlid fish Oreochromis niloticus,at different salinity levels

ABSTRACT

The effects of cadmium concentration (0, 2.5 and 5 mg L^?1) on melanocyte-stimulating hormone (MSH), melanophore index (MI), and melanophore number (MN), as well as a microscopic examination of scale melanocytes in tilapia (Oreochromis niloticus Linnaeus, 1757) was evaluated at different salinity levels (0, 5 and 15 ppt). The levels of MSH, MI, and MN were lower in Cd-exposed fish than in control fish (not exposed to Cd) at salinity level of 0 and 5 ppt. In ppt, however these levels of MSH, MI and MN in control and Cd-exposed fish were not significantly different. In the media without Cd, the levels of MSH, MI and MN were not significantly different at all salinities. The morphological changes of melanophores were higher in Cd-exposed fish than in control fish at salinity 0 and 5 ppt, respectively. These morphological changes were not significantly different in the control fish at all salinities as well as in fish exposed to 0–5 mg L^?1 Cd at salinity of 15 ppt. This study therefore demonstrates that the toxic effect of Cd on MSH levels and melanophore morphology decreased with increasing salinity. Further, due to the sensitivity of chromatophores to Cd, melanophore morphology is proposed as a biomarker of Cd exposure in aquatic ecosystems.