Increased efficiency of homologous recombination in ES cells by cleavage at both ends of homology in the targeting vector

Transgenic Research -     

The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1–871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1–RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1–RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1–RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.     

Differential effects of abscisic acid on desiccation tolerance and carbohydrates in three species of liverworts

Tissues of three species of in vitro grown liverworts, Riccia fluitans, Pallavicinia lyellii, and Marchantia polymorpha, were subjected to rapid drying with and without preculture for 1 week on medium containing 10 μM ABA. ABA preculture initiated total desiccation tolerance in R. fluitans, whereas control tissues were killed after 30 min of drying. Survival was also improved in P. lyellii, whereas ABA did not affect survival of M. polymorpha after rapid drying. ABA treatment did, however, reduce the rate of water loss in M. polymorpha. Total soluble carbohydrates were increased in ABA-treated R. fluitans and P. lyellii, but not in M. polymorpha, although there was no correlation between survival and changes in the percentage of these carbohydrates as reducing sugars. These differences in response to ABA and desiccation likely reflect different adaptations of these three species to conditions in situ.     

Tumor-host interactions contribute to the elevated expression level of alpha1-antichymotrypsin in metastatic breast tumor xenografts

We investigated alpha1-antichymotrypsin (ACT) gene expression in xenograft tumors generated by two isogenic human breast cancer cell lines derived from the same parent, MDA-MB-435, which display opposite metastatic behaviors. Microarray and real-time PCR experiments showed an overexpression of this serine protease inhibitor in the metastatic tumors (M-4A4T) and its derived metastases (M4-Mets) compared with the weakly metastatic tumors (NM-2C5T), and its release into the blood was confirmed by western-blotting. However, functional assays in vivo using genetically engineered tumor cells demonstrated that ACT up-regulation was not, by itself, responsible for the metastatic phenotype. We also made observations that ACT gene regulation was sensitive to tumor-host interactions: inoculation of these lines into the mouse mammary gland greatly increased ACT production and accentuated the intrinsic difference observed when they are cultured in vitro. Sensitivity of tumor cells to their environment was further analyzed by in vitro experiments, which demonstrated that a purified ECM environment and soluble components from normal host mammary cells were both able to significantly promote ACT expression. In addition, we took advantage of the xenogeneic nature of the model to measure ACT expression by the host cells (mouse) and the tumor cells (human) within the neoplasm using species-specific primers in real-time PCR experiments. It was found that the presence of tumor cells, irrespective of their metastatic capabilities, induced local ACT production by host cells at the primary and secondary tumor sites. Thus, this work indicates that there is a specific association of ACT overexpression with the metastatic phenotype in our breast cancer metastasis model. Moreover, because of the xenogeneic nature of our system, we were able to provide evidence of tumor-host reciprocal regulation of ACT production.     

Molecular cloning and tissue distribution of microsomal-1 and cytosolic prostaglandin E synthases in macaque

Prostaglandins derived from arachidonic acid are involved in a wide variety of physiological and pathological processes. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases. These enzymes have been identified in human, but only partially in the monkey where microsomal PGES-1 and cytosolic PGES have not been characterized. The present study was undertaken to clone these enzymes and to study their tissue distribution, along with mPGES-2. The coding sequence of Macaque mPGES-1 is 98% homologous to human mPGES-1 at the nucleic acid level and the deduced amino acid sequence has 98% homology with the human protein. The Macaque cPGES cDNA is more than 99% homologous to the human and the deduced amino acids sequence is identical to that of the human cPGES. By Northern blot analysis, we found that mPGES-2 and cPGES mRNA were expressed in the endometrium, myometrium, ovary and oviduct, albeit at different levels, while mPGES-1 mRNA was detected at a weak level, mainly in the oviduct. Western Blot analysis revealed that mPGES-2, mPGES-1 and cPGES proteins were present in all tissues tested. These results suggest that production of PGE2 in Macaque may involve more than one PGES and that further studies will be needed to fully understand the conditions under which each PGES contributes to PGE2 production.     

Antagonist Microorganisms with the Ability to Control <Emphasis Type="Italic">Pythium</Emphasis> Damping-off of Tomato Seeds in Rockwool

A total of 237 microorganisms were isolated from five different greenhouse tomato growing media. Of those, 40 microorganisms reduced the in vitro mycelial growth of both Pythium aphanidermatum and Pythium ultimum. The ability of these microorganisms to control damping-off was then tested in rockwool. As a result, Pseudomonas corrugata strains 1 and 3, Pseudomonas fluorescens subgroup F and G strains 1, 2, 3, 4 and 5, Pseudomonas marginalis, Pseudomonas putida subgroup B strain 1, Pseudomonas syringae strain 1 and Pseudomonas viridiflava significantly reduced damping-off caused by P. ultimum or P. aphanidermatum. Pseudomonas marginalis was the only microorganism that significantly reduced damping-off caused by both pathogens.