Genetic variability for esterase enzyme in Onobrychis species

Summary Understanding polymorphism at the enzyme level is basic to its use in population and genetic studies. However, no such information is available on the variability among different sainfoin (Onobrychis) species. Therefore, our objective was to study the existence of genetic polymorphism for esterase in 17 Onobrychis species and three cultivars of O. viciifolia Scop. Three regions of banding were observed in all the materials tested, with the number of bands varying from 0 to 3, 3 to 14, and 1 to 2 bands in each of these zones, which have been designated EST1, EST2, and EST3 respectively. All the materials studied had unique banding patterns, the only common feature being that all of them, except one species, had isozyme 1. Identification was possible only for four species (O. iberica, O. kachetica, O. transcaucasica, and O. bieberstenii) and one cultivar (Nova) based on the banding patterns. Large diversity was evident from the wide range of percent similarity values (0%–79%). Subsequent studies should be directed in using these isozyme banding patterns as markers to the desirable agronomic and quality traits of different germplasm lines.This work was supported by USDA Specific Cooperative Agreement No. 58-7MN1-8-143 from the Plant Stress and Water Conservation Unit, USDA-ARS, Lubbock, Texas. Joint contribution of the Texas Tech University, Lubbock, Texas and the USDA-ARS. TTU Journal no. T-4-302     

Structural prediction and analysis of VIH-related peptides from selected crustacean species

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.     

Targeted Methylation of the Epithelial Cell Adhesion Molecule (EpCAM) Promoter to Silence Its Expression in Ovarian Cancer Cells

The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.     

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (<Emphasis Type="Italic">Populus tremula</Emphasis> L.)

Key message

Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed.

Abstract

Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.