Detection of self-complementary inverted repeats by single forward primer driven PCR

Inverted repeat gene structures designed for silencing functional genes have been widely used both in academic and applied research. The correct orientations of such structures are usually validated with restriction analysis and/or sequencing. We speculated that the inverted repeat nature of such constructs can be shown by a simple PCR reaction with a single forward primer. To test this hypothesis five different constructs were established from grapevine sequences in a hairpin-intron style silencing system. We were able to amplify the appropriate products in each case. Thus a forward-primed PCR alone may be sufficient to prove the inverted repeat nature of the desired constructs

Detection of self-complementary inverted repeats by single forward primer driven PCR

Inverted repeat gene structures designed for silencing functional genes have been widely used both in academic and applied research. The correct orientations of such structures are usually validated with restriction analysis and/or sequencing. We speculated that the inverted repeat nature of such constructs can be shown by a simple PCR reaction with a single forward primer. To test this hypothesis five different constructs were established from grapevine sequences in a hairpin-intron style silencing system. We were able to amplify the appropriate products in each case. Thus a forward-primed PCR alone may be sufficient to prove the inverted repeat nature of the desired constructs

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

Abstract The grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.</jats:p

The Uncoupling of the Effects of Formins on the Local and Global Dynamics of Actin Filaments

In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled