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Query: UNIPROT:P06889 (Mol)
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Pantropic retroviral vectors were used to introduce transgenes into Japanese medaka (Oryzias latipes). These vectors contain the long terminal repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous sarcoma virus (RSV). Because these pseudotyped retroviral vectors contain the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammalian species. Newly fertilized medaka eggs (intact or dechorionated) were electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pantropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substantially affect the survival rate of embryos. PCR amplification demonstrated proviral sequences in up to 60% of the 2-month-old fish. The efficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retroviral vectors without electroporation also resulted in proviral integration in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction (PCR) as positive F1 reaction animals confirmed the integration of a single copy of the provirus into the host genome. Three P1 transgenic females transmitted the proviral sequence to 50% of their F1 progeny in a back cross with wild-type males, suggesting that the entire germline of these P1 fish was transformed by the pantropic retroviral vector. Expression of the neomycin phosphotranferase transgene in F1 transgenic individuals was detected by reverse transcription (RT)-PCR amplification of the neo mRNA sequence. Furthermore, expression of a beta-galactosidase transgene was also observed in 4-day-old F1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient method to stably introduce and express foreign genes in medaka.
Mol Mar Biol Biotechnol 1997 Dec
PMID:Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes). 941 87

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus-hemagglutinin (HA) and vesicular stomatitis virus-G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.
Mol Biol Cell 1998 Mar
PMID:An apical-type trafficking pathway is present in cultured oligodendrocytes but the sphingolipid-enriched myelin membrane is the target of a basolateral-type pathway. 948 29

Several investigators have recently examined the effect of Fas (CD95)-mediated apoptotic cell death on target cells (TC). The effect of Fas-mediated death on viral RNA within the TC, however, has not been explored. In this study, we investigated the ability of the Fas pathway to mediate pre-lytic degradation of vesicular stomatitis virus (VSV) RNA and TC RNA. We show that engagement of Fas antigen on VSV-infected Jurkat cells induces pre-lytic degradation of VSV RNA transcripts, whereas full-length VSV genome RNA, known to be tightly associated with viral proteins, is not degraded. Cellular RNA, including beta-actin and glyceraldehyde-3-phosphate-dehydrogenase mRNAs, is also degraded by Fas-mediated cytotoxicity. In addition, Fas-mediated cytotoxicity reduced the yield of VSV plaque-forming units (PFU) from Jurkat by an average of 82.0%. An anti-Fas blocking Ab inhibited the RNA degradation and restored the number of VSV PFU to near control levels. These data indicate that the Fas lytic pathway could play a role in the elimination of viruses through degradation of intracellular viral RNA. reserved
Mol Immunol 1997 Oct
PMID:Fas-mediated cytotoxicity induces degradation of vesicular stomatitis virus RNA transcripts and reduces viral titer. 951 64

The control of insects that transmit disease and damage crops has become increasingly difficult. The ability to genetically engineer insects would facilitate strategies to protect crops and block arthropod vector-borne disease transmission. Transformation vectors based on insect transposable elements have been developed, but most have limited host ranges. A promising alternative is the pantropic retroviral vector, which is packaged with the envelope glycoprotein from vesicular stomatitis virus and is replication-defective. We show here that pantropic murine retroviral vectors can mediate high-level expression of foreign genes in somatically transformed insect larvae and adults of three dipteran genera. This success demonstrates the potential for germline transformation mediated by pantropic retroviral vectors.
Insect Mol Biol 1998 Aug
PMID:Pantropic retroviral vectors mediate somatic cell transformation and expression of foreign genes in dipteran insects. 966 70

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum-to-Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.
Mol Biol Cell 1998 Nov
PMID:Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. 980 9

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.
Mol Immunol 1998 Jul
PMID:Point mutations in the beta chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I/peptide complex over a broad interface area. 982 58

A soluble factor which augments the expression of major histocompatibility complex class I (MHC I) antigens on a number of murine tumor cell lines, has been isolated from the culture supernatants of mixed lymphocyte reaction of spleen cells derived from C57B1/6, Balb/c and Swiss mice. The factor, termed MHC-augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography and reverse phase HPLC. MHC-AF activity is associated with an 18 kDa molecule. MHC-AF activity was resistant to pH 2.0 treatment and partially purified MHC-AF preparations did not have any activity in L929 cell/vesicular stomatitis virus (VSV) interferon bioassay system. Antibodies to IFN-gamma did not block the activity of MHC-AF. These results indicate that a MHC-AF distinct from IFN-gamma, is produced by mouse spleen cells undergoing a mixed lymphocyte reaction.
Exp Mol Med 1998 Jun 30
PMID:Partial purification and characterization of a novel murine factor that augments the expression of class I MHC antigens on tumor cells. 987 29

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.
Mol Biol Cell 1999 Jan
PMID:Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle. 988 Mar 32

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15 degreesC is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER-Golgi transport.
Mol Biol Cell 1999 Feb
PMID:Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment. 995 Jun 87

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.
J Mol Biol 1999 Apr 02
PMID:Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells. 1009 52


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