Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells.

Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 microM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 microM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

Interaction of fusogenic peptides with an antisense oligonucleotide in solution and in the presence of micelles: conformational studies.

The conformational effect of the interaction between various fusogenic peptides and an 18mer single stranded antisense oligonucleotide (ODN), targeted towards the green fluorescent protein mRNA, has been studied by circular dichroism spectroscopy in water and in the presence of anionic lysolipid micelles. The peptides used were the third helix of Antennapedia homeodomain pAntp-(43-58), the flock house virus FHV-gamma-(364-407) peptide, and its N-terminal gamma1-(364-384) and C-terminal gamma2-(390-407) fragments. The most significant conformational changes were observed in ODN-pAntp-(43-58) and ODN-FHV-gamma2 complexes. The pAntp-(43-58) forms a complex with ODN through electrostatic interaction resulting in profound changes in the conformation of both the peptide and the ODN. In the case of FHV-gamma2 peptide the complex formation takes place without altering the structure of ODN, and the decreased ratio of deltaepsilon208/deltaepsilon222 reflects the insertion of the complexed peptide into the micelle.

Use of antisense oligonucleotides to verify the role of the alpha(1A)-adrenergic receptor in the contractility of the rat uterus post partum.

The adrenergic system plays a major role in the regulation of the contractility of the uterus during pregnancy. This study investigated the role of the alpha(1A)-adrenergic receptor (AR) in this regulation. The use of partial phosphorothioate antisense oligodeoxynucleotides (AONs) permitted the sequence-selective inhibition of AR gene expression. AONs were injected together with a cationic liposomal carrier agent into the post partum rat uterus. Incubation for 12 or 24 h with the most effective AON (480-AON) caused a 58.7 or 53.0% inhibition, respectively, of the expression of the alpha(1A)-AR density, whereas incubation for 36 or 48 h resulted in only a 38.8 or 26.7% inhibition, respectively. The decrease of the alpha(1A)-AR density by 480-AON was demonstrated by Western blot analysis and a radioreceptor binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus after AON treatment were measured on isolated rat uterine tissue by electric field stimulation. The significant decrease in the ability of the uterus to contract was indicated by the area under the curve method. The electric field studies revealed that the specific alpha(1A)-blockers 5-methylurapidil and WB 4101 inhibited the rhythmic contraction by about 74 and 70% in the control uteri and by 25 and 20% in 480-AON-treated uteri, respectively. The curves for the beta-mimetic (terbutaline) and alpha(1D)-antagonist (BMY7370) inhibitors were unchanged after 480-AON treatment of the uteri. These results suggest the importance of the alpha(1A)-AR in the tocolytic effect exerted by the alpha(1)-antagonist, although high concentrations of antagonists can not exclude the role of alpha(1D)-ARs, too. Additionally, these prove that the knockdown transformation by AONs offers a useful animal model for the investigation of receptors controlling the function of uterine tissue.

[Pharmacological models to investigate the role of the adrenergic system on post-partum rat uterus]. / Farmakológiai modell az adrenerg rendszer szerepének vizsgálatára post partum patkány uteruson.

The adrenergic system plays a major role in the regulation of the pregnant uterine contractility. Our aim was to develop an experimental animal model to study the role of the alpha 1A-adrenergic receptor (AR) in uterine motor activity by antisense oligonucleotides (AONs). AONs were injected with DOTAP and pluronic gel into the uterine lumen of post-partum rats 2-3 hours after delivery. The decrease of the alpha 1A-AR density by AON was demonstrated by RT-PCR method, Western blot analysis and radioligand binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus were measured on isolated rat uterine tissue by electric field stimulation (EFS). The EFS investigation demonstrated that the effect of the specific alpha 1A-blocker 5-methylurapidil and WB4101 was significantly decreased in the AON-treated rat uterus as compared to the control group but the effect of the beta-mimetic terbutalin and alpha 1D-antagonist BMY7378 was unchanged. Our result suggest that the alpha 1A-ARs play a very important role in the regulation of uterine contractility, and may serve as the basis for a subsequent new group of tocolytics (uterus selective alpha 1-antagonists), which may lead to more selective therapy than currently used beta-mimetics.

Immunolocalization of a novel annexin-like protein encoded by a stress and abscisic acid responsive gene in alfalfa.

We report here on the isolation and characterization of a full-length cDNA clone from alfalfa termed AnnMs2 encoding a 333 amino acid long polypeptide that shows 32-37% sequence identity with both mammalian and plant annexins, and has four tandem repeats. While other plant annexins exhibit a high level of sequence similarity to each other (up to 77% identity at amino acid level), AnnMs2 appears to be a distinct type of plant annexins. All the four endonexin folds contain the conserved eukaryotic motif within this alfalfa protein, but this element is considerably different in the second repeat. The AnnMs2 gene is expressed in various tissues of alfalfa with elevated mRNA accumulation in root and flower. This gene is activated in cells or tissues exposed to osmotic stress, abscisic acid (ABA) or water deficiency. The recombinant AnnMs2 protein is able to bind to phospholipid in the presence of Ca2+. Indirect immunofluorescence studies using affinity purified rabbit anti-AnnMs2 peptide antibody show mainly nucleolar localization, but the protein sequence lacks the usual nuclear localization signal. The potential role of this novel annexin-like protein in the basic and stress-induced cellular functions is discussed.

Reduction of mispriming in amplification reactions with restricted PCR.

A simple method is described for reducing nonspecific background, which is caused by mispriming during PCR. Besides the standard pair of primers, 3'-dideoxy-terminated competitor oligonucleotides were added to the amplification. Sequences to those of the primers which had identical base. In this way enhanced specificity was achieved. The competitor oligonucleotides may act by masking possible sites of nonspecific primer-template interaction, thus excluding undesired chain extensions. This technique is generally applicable when highly degenerate primers are used and therefore expands the potential of "restricted" PCR.

Prokaryotic test system for evaluation of oligonucleotide-affected antisense inhibition.

A new test system is introduced for the estimation of the antisense effect of various types of oligodeoxy-nucleotides. This system is based upon the inhibition of translation of P2 phage repressor protein. Evidence for a direct correlation between phage yield and antisense inhibition was obtained by comparing the concentration-dependent effectiveness of an antisense phosphorothioate 18-mer, its mutated version, and the unmodified oligonucleotide. Oligonucleotides of complementary sequences were synthesized against the ribosome binding site and different sites of the coding region. In our test system only small differences were found based on the location of the target sequence. Rather, the degree of inhibition was related to the calculated ability to hybridize based on Tm values. In this way the sensitivity of the test system could also be demonstrated.