Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector

12 pages, 5 figures, 3 tables.A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells.The support of the European Molecular Biology Laboratory during the initial phases of this work is acknowledged. S.F. was supported by the Swedish Medical Research Foundation, and A.M. was supported by the Juan March Foundation. Grants were obtained from the Swedish Cancer Society, the Wallenberg Foundation, the Children's Cancer Fund, and Kjell and Marta Beijer's Foundation.Peer reviewe

Molecular characterization of an anther-specific gene from tobacco shows sequence similarity to a tapetum-specific gene from tomato

We have cloned and determined the DNA sequence of the cDNA of ntGRP15 . The cDNA ntGRP15 represents an anther-specific, developmentally regulated gene from Nicotiana tabacum that encodes a glycine-rich protein. Northern analysis shows that the gene is specifically expressed in anthers and is stringently regulated during anther development. It appears only in anthers at the meiosis to free microspore stages of development. The encoded protein is small (12.2 kDa), has a 31% glycine content and contains a putative signal sequence. By both nucleotide and amino acid sequence alignment, the gene shows high sequence similarity to a gene previously isolated from Lycopersicon esculentum, namely, TomA92b9 . High glycine content, presence of a signal sequence and similarity to the tomato TomA92b9 gene suggests the protein functions as a structural cell wall protein, possibly involved in pollen exine formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42313/1/497-12-4-250_00120250.pd

Continued Neurogenesis in Adult Drosophila as a Mechanism for Recruiting Environmental Cue-Dependent Variants

Background The skills used by winged insects to explore their environment are strongly dependent upon the integration of neurosensory information comprising visual, acoustic and olfactory signals. The neuronal architecture of the wing contains a vast array of different sensors which might convey information to the brain in order to guide the trajectories during flight. In Drosophila, the wing sensory cells are either chemoreceptors or mechanoreceptors and some of these sensors have as yet unknown functions. The axons of these two functionally distinct types of neurons are entangled, generating a single nerve. This simple and accessible coincidental signaling circuitry in Drosophila constitutes an excellent model system to investigate the developmental variability in relation to natural behavioral polymorphisms. Methodology/Principal Findings A fluorescent marker was generated in neurons at all stages of the Drosophila life cycle using a highly efficient and controlled genetic recombination system that can be induced in dividing precursor cells (MARCM system, flybase web site). It allows fluorescent signals in axons only when the neuroblasts and/or neuronal cell precursors like SOP (sensory organ precursors) undergo division during the precedent steps. We first show that a robust neurogenesis continues in the wing after the adults emerge from the pupae followed by an extensive axonal growth. Arguments are presented to suggest that this wing neurogenesis in the newborn adult flies was influenced by genetic determinants such as the frequency dependent for gene and by environmental cues such as population density. Conclusions We demonstrate that the neuronal architecture in the adult Drosophila wing is unfinished when the flies emerge from their pupae. This unexpected developmental step might be crucial for generating non-heritable variants and phenotypic plasticity. This might therefore constitute an advantage in an unstable ecological system and explain much regarding the ability of Drosophila to robustly adapt to their environment

Induction of G0/G1 cell cycle arrest in ovarian carcinoma cells by the anti-inflammatory drug NS-398, but not by COX-2-specific RNA interference

Cyclooxygenases, particularly COX-2, play an important role in tumor development and progression. We have previously shown that COX-2 expression is an independent prognostic factor in human ovarian carcinoma. In this study, we investigated the effects of the inhibition of COX isoforms by the NSAID NS-398 as well as by COX-isoform-specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1 and an induction of high levels of COX-2 and PGE2 after stimulation with interleukin-1{beta}. In contrast, SKOV-3 cells were negative for both COX isoforms. In OVCAR-3 cells, PGE2 production was inhibited by NS-398 in concentrations of 1 {my}M and by a COX-2-specific silencing RNA (siRNA), while a COX-1-specific siRNA did not have an effect. This suggests that COX-2 is the major source of PGE(2) in this cell line. To dissociate COX-2-specific and non-COX-2-specific effects on cell proliferation, a proliferation assay was performed after incubation of cells with NS-398 and COX siRNAs. NS-398 induced an inhibition of cell proliferation at concentrations of 50–500 {my}M, which are above the concentrations needed for the inhibition of PGE2 production. This inhibitory effect was present in the COX-positive cell line OVCAR-3 as well as in the COX-negative cell line SKOV-3 and could not be reverted by addition of exogenous PGE2. Neither COX-1- nor COX-2-specific siRNAs had an effect on cell proliferation of OVCAR-3 cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with NS-398, but not with COX siRNAs. These experiments suggest that NS-398 reduced cell proliferation in ovarian carcinoma cells by induction of G0/G1 cell cycle arrest independent of COX-2 inhibition. Our study shows that specific inhibition of COX isoforms by RNAi could be used to dissociate effects of NSAIDs. Furthermore, our results suggest that cell cycle arrest is one of the primary mechanisms responsible for the antiproliferative effects of NS-398 on ovarian carcinoma cells

Abstrakt, a DEAD box protein, regulates Insc levels and asymmetric division of neural and mesodermal progenitors

AbstractAsymmetric cell division generates cell diversity in bacteria, yeast, and higher eukaryotes [1–3]. In Drosophila, both neural and muscle progenitors divide asymmetrically [4–16]. In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation. Abstrakt (Abs) is a DEAD-box protein that regulates aspects of cell polarity in oocytes and embryos [17]. We use a conditional allele of abs to investigate its role in neural and muscle progenitor cell polarity. In neuroblasts we observe loss of apical Insc crescents, failure in basal protein targeting, and defects in spindle orientation. In the GMC4-2a cell we observe loss of apical Insc crescents, defects in basal protein targeting, and equalization of sibling neuron fates; muscle precursors show a similar equalization of sibling cell fates. These phenotypes resemble those of insc mutants; indeed, abs mutants show a striking loss of Insc protein levels but no change of insc RNA levels. Furthermore, we find that the Abs protein physically interacts with insc RNA. Our results demonstrate a novel role for Abs in the posttranscriptional regulation of insc expression, which is essential for proper cell polarity, spindle orientation, and the establishment of distinct sibling cell fates within embryonic neural and muscle progenitors

Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector.

A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells