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PubMed1993

Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.

Aging↗
PubMed1996

Expression of oncofetal fibronectin in porcine conceptuses and uterus throughout gestation.

Oncofetal fibronectin is reportedly expressed specifically by trophoblast tissue and some tumours and speculated to mediate placental attachment. In the present study, a monoclonal antibody (FDC-6) to human oncofetal fibronectin was used to characterize temporal and spatial changes in the expression of oncofetal fibronectin at the fetal-maternal interface of pigs. Conceptus and uterine tissues were collected from gilts throughout normal pregnancy and processed for immunohistochemical characterization. Results indicated that oncofetal fibronectin was constitutively expressed by both porcine conceptus and uterus throughout gestation. The most abundant staining for oncofetal fibronectin was found in the allantochorion and detectable levels of oncofetal fibronectin were also detected in luminal and glandular epithelial cells in the uterus. During the second-half of pregnancy, oncofetal fibronectin was also detected in fibroblast-like cells in the uterine stroma, but not in the stroma of the allantochorion. Owing to the non-invasive nature of the porcine placenta, the abundant expression of oncofetal fibronectin by the trophoblast and uterus may influence attachment between chorion and endometrium during pregnancy. Since attachment is the first step in implantation and placentation in all mammalian species, the pig may represent an excellent animal model to study interactions between trophoblast and endometrium mediated by oncofetal fibronectin.

Animals↗
PubMed1990

High levels of c-fos proto-oncogene expression in normal human adult skin.

The proto-oncogene c-fos is thought to play an important role in the modulation of cell growth and differentiation. In normal tissues that have been studied to date, c-fos expression has been found to be regulated in a tissue-specific manner. Actually, little is known about its expression in normal human adult skin (NHAS). Moreover, the epidermis is a useful tissue to study the role of cellular oncogenes because keratinocytes can be observed simultaneously in their proliferative as well as differentiated state. We studied c-fos expression in NHAS using different molecular approaches which permit us to characterize and localize c-fos products within the epidermis, specifically, at the RNA level by Northern blot and in situ hybridization, and at the protein level by immunofluorescence and Western blotting. Here, we show that both c-fos mRNA and protein are present at high levels in NHAS. These results contrast with the low level of c-fos expression reported for most human adult tissues. Furthermore, c-fos expression is visible throughout the epidermal layers indicating that it is not restricted to proliferating basal cells. The epidermis, therefore, represents the first human adult tissue where c-fos is expressed at high levels in vivo and provides an interesting model to further elucidate the role of this proto-oncogene in normal and pathologic conditions.

Blotting, Northern↗
PubMedDate not supplied

Expression of the intermediate filament peripherin in skin tumors.

Peripherin is a neuronal intermediate filament reportedly expressed in neuroendocrine skin carcinomas and some melanocytic tumors. In order to assess the diagnostic usefulness of antibodies to peripherin in dermatopathology, we studied its expression in 68 skin tumors mostly of neural origin, and compared this expression with that of neurofilaments, a well-established neuronal marker. Antibodies to peripherin and neurofilaments both labeled dermal neurons present in normal skin and within the tumors. Specific labeling of tumor cells for peripherin was seen in only 2/5 neuroendocrine skin carcinomas, 3/18 benign nevi and 7/19 melanomas; even in these cases the percentage of tumor cells did not exceed 20%. We conclude that antibodies to peripherin may be used for the demonstration of cutaneous neurons in normal and diseased skin; however their diagnostic usefulness appears limited, less than that of antibodies to neurofilaments.

Humans↗
PubMed1996

Novel, high expressing and antibiotic-controlled plasmid vectors designed for use in gene therapy.

The promise of effective gene therapy can only be accomplished by high-level expression and regulatable delivery of gene products. To achieve this end, a eukaryotic expression plasmid was modified to make transcription dependent on a tetracycline(Tc)-regulated chimeric transactivator. Mouse muscle injected with this two plasmid cis/trans control system expressed reporter proteins at levels five- to 10-fold greater than the cytomegalovirus immediate-early promoter-controlled parental plasmid. Tetracycline could be useful to either repress or activate transactivator-controlled expression based on the position of the tetO control sequences within the reporter plasmid. Finally, a prototype single plasmid construct was made and shown to express a self-regulating bicistronic transcript containing both the reporter and the transactivator. These Tc-controlled plasmids, termed maximum expression and regulated vectors (MERVs), have the potential to target a variety of gene therapy applications.

Animals↗
PubMed1996

Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue.

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules. In vivo, in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers [> or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100-1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.

Adenoviridae↗
PubMed1989

Posttranscriptional trans-activation in cauliflower mosaic virus.

The ability of plant cells to translate dicistronic mRNAs that mimic a segment of the polycistronic 35S RNA from cauliflower mosaic virus has been tested. The chloramphenicol acetyltransferase and beta-glucuronidase open reading frames (ORFs) were fused in-frame to the second viral cistron (ORF I). Efficient reporter expression from the corresponding plasmids in plant protoplasts was observed only upon cotransfection with viral DNA. The trans-activating gene maps at ORF VI, which is expressed from a separate, monocistronic messenger (19S RNA). Deletion analysis shows that trans-activation selectively enhances downstream gene expression; the high expression of the upstream ORF is not further increased. The major reporter transcript remained bicistronic upon trans-activation, and its abundance varied only to a limited extent. Results indicate that trans-activation enhances the translation of downstream ORFs on polycistronic mRNAs derived from cauliflower mosaic virus.

Base Sequence↗
PubMed1993

Recombinant expression and partial characterization of the human formyl peptide receptor.

FMLP-receptor DNA was expressed in Escherichia coli. The expressed product could specifically bind FMLP. This is the first-reported expression of a functional FMLP receptor in Escherichia coli. We confirm that receptor glycosylation is not essential for ligand binding. A deletion mutant did not bind FMLP, suggesting that the deleted portion plays a role in ligand binding.

Amino Acid Sequence↗
PubMed1998

In vivo effects of mutations in woodchuck hepatitis virus enhancer II.

Woodchuck hepatitis virus (WHV) enhancer II (EnII) is located upstream of the major pregenomic RNA promoter and is thought to play an important role in the insertional activation of the N-myc2 gene during WHV hepatocarcinogenesis. WHV EnII is recognized by at least three host transcription factors: HNF-1, HNF-4, and Oct-1. Here, the roles of these EnII-binding factors in viral transcription and replication have been further examined. In HepG2 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) gene whose expression is dependent upon EnII, mutations in either the HNF-1 or the HNF-4 site strongly reduced CAT activity, while ablation of the Oct-1 site decreased CAT expression only twofold. Mutations in more than one site completely abolished reporter expression. These same mutations were also tested in an overlength WHV genome for their impact on viral replication and gene expression. In transfected HepG2 cells, lesions in the HNF-1 site inactivated pregenomic RNA expression and viral reverse transcription, with only minimal effects on the expression of other viral mRNAs. By contrast, Oct-1 site lesions had no effect on either viral RNA synthesis or DNA replication, and HNF-4 site lesions produced a modest reduction of pregenomic RNA but had no impact on viral DNA synthesis. Testing of the mutants in susceptible woodchucks revealed that, as expected, viruses with lesions in the HNF-1 site were nearly noninfectious, while mutants with lesions at the Oct-1 site were fully replication competent. HNF-4 site mutants were replication competent but may display reduced levels of replication in the intact animal host. We conclude that (i) EnII is primarily devoted to the regulation of pregenomic RNA in WHV, (ii) HNF-1 is essential for EnII function in vivo, and (iii) HNF-4 plays a demonstrable but adjunctive role in EnII function.

Animals↗
PubMed1997

A ubiquitin-conjugating enzyme is essential for developmental transitions in Dictyostelium.

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for approximately 10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

Amino Acid Sequence↗
PubMed1998

Expression of active thrombopoietin and identification of its key residues responsible for receptor binding.

In this report expression of the biologically active N-terminal half (amino acids 1-153) of thrombopoietin (TPO153) in Escherichia coli is described and the structure-function relationships in TPO are explored. TPO153 was chosen for expression because of its full biological activity. Since natural TPO153 cDNA expressed poorly, synthetic cDNA was constructed with a unique polymerase chain reaction to enhance the expression. In addition, the 5'-end codons of the synthetic cDNA were altered to maximize the expression. The expressed TPO153 was refolded and then purified to homogeneity. The protein is biologically active, and interestingly, the EC50 of this protein is 8-10-fold smaller in a TPO-dependent cell proliferation assay than that of full-length wild-type TPO. In order to identify the amino acid residues that are involved in the interaction between TPO and its receptor, all charged residues and some of the uncharged residues on the four putative helices of TPO were mutated and biological activities of the mutant proteins were examined. The mutagenesis studies suggest that there are at least two clusters of residues that are vital for TPO to be able to interact with its receptor. These residues are centred respectively around arginine 10 on helix 1 and around lysine 138 on helix IV. The successful expression of the protein in E. coli will greatly facilitate biochemical and crystallographic studies of TPO, and the structure-function relationship studies suggest that TPO has two binding sites which may interact with two individual receptors, resulting in dimerization of the receptors.

Amino Acid Sequence↗
PubMed1993

Expression of the EVI1 gene in chronic myelogenous leukemia in blast crisis.

The EVI1 gene encodes a nuclear, zinc finger, DNA binding protein expressed after provirus insertion into the Fim-3 or EVI1 loci in murine leukemia myeloid cells. EVI1 is also expressed by cells from AML patients with chromosome rearrangements involving band 3q26, the putative location of the EVI1 gene, but expression of this gene is not detected in normal bone marrow. We report expression of EVI1 by cells from a patient with chronic myelogenous leukemia in blast crisis (CML/BC) whose cells showed inv(3)(q22q26). In vitro culture of these cells resulted in macrophage differentiation and loss of EVI1 expression. Results in this patient suggest EVI1 expression played a role in CML blast transformation. Patients with CML/BC and other nonrandom chromosome abnormalities involving chromosome 3q26 should be evaluated for EVI1 expression.

Adult↗
PubMed1996

Isolation, characterization, and expression of Le-msx, a maternally expressed member of the msx gene family from the glossiphoniid leech, Helobdella.

The msx gene family is one of the most highly conserved of the nonclustered homeobox-containing genes. We have isolated an msx homolog (Le-msx) from the glossiphoniid leech, Helobdella robusta, and characterized its pattern of expression by whole mount in situ hybridization. In situ expression and reverse transcription polymerase chain reaction (RT-PCR) data results show that Le-msx is a maternal transcript initially uniformly distributed in the cortex of immature oocytes that becomes asymmetrically localized to the polar regions of the uncleaved zygote. This is the earliest reported expression for the msx gene family and the first maternally expressed homeodomain-containing transcription factor reported in annelids. During embryonic development, Le-msx is expressed in all 10 embryonic stem cells and their segmental founder cell descendants. At midembryonic stages, Le-msx is expressed in the expanding germinal plate. Le-msx is confined to the central nervous system and nephridia at late (stage 9) stages and subsequently disappears from nephridia. In addition, we present a phylogenetic hypothesis for the evolution of the msx gene family, including the identification of a putative C. elegans msx homolog and the realignment of the sponge msx homolog to the NK class of homeodomain genes.

Amino Acid Sequence↗
PubMed2026

Suppression of AAV-Delivered Transgene Expression Using Artificial MicroRNAs Delivered by an Alternative AAV Serotype.

Adeno-associated virus (AAV) gene transfer vectors mediate long-term expression in nondividing cells, an advantage for treating chronic disorders. However, current platforms lack a way to selectively shut down transgene expression if adverse effects arise. To create an "off switch," we hypothesized that incorporating unique artificial microRNA (amiRNA) target sequences into an AAV expression cassette would allow subsequent suppression of transgene expression using a second AAV vector encoding the cognate amiRNA. We introduced 22-nt sequences absent from human and mouse transcriptomes into the 3' untranslated region (UTR) of a therapeutic AAV cassette. To identify optimal amiRNAs, two tandem copies of each amiRNA were cloned into the 3'UTR of an mCherry reporter gene. In vitro assessment of six amiRNA/target pairs using a dual luciferase assay identified four amiRNAs that efficiently suppressed reporter expression. Cells cotransfected with target site 3 (TS3) and amiRNA-T3B showed the greatest reduction in luciferase activity (80%, p < 0.0001) and were selected for further study. The "off-switch" system was then evaluated using an AAV5 therapeutic vector expressing a recombinant humanized anti-IgE monoclonal antibody (AAV5-TBG-anti-IgE-TS3), designed for long-term suppression of allergen-induced reactions. Co-transfection of HEK293T cells with anti-IgE-TS3 and amiRNA-T3B significantly reduced anti-IgE mRNA and protein levels relative to a control amiRNA (p < 0.0001). In vivo testing in Balb/c mice (n = 5) involved intravenous administration of AAV5-anti-IgE-TS3 (3.2 &#xd7; 1010 gc), followed 4 weeks later by an AAVrh.10 amiRNA vector (AAVrh.10-TBG-amiRNA-T3B; 1 &#xd7; 1011 gc). Control mice receiving only the therapeutic vector expressed 18.4 &#xb1; 13.8 &#xb5;g/mL serum anti-IgE at 10 weeks. In contrast, mice receiving the amiRNA "off" vector showed marked suppression of anti-IgE (0.3 &#xb1; 0.15 &#xb5;g/mL, p < 0.0001). These findings provide proof-of-concept that AAV-delivered amiRNAs can selectively switch off transgene expression, offering a strategy to improve the safety of AAV-mediated gene therapies.

Dependovirus↗
PubMed1998

Candida albicans HEX1 gene, a reporter of gene expression in Saccharomyces cerevisiae.

The Candida albicans HEX1 gene was examined as a reporter of gene expression in Saccharomyces cerevisiae. The galactose-inducible S. cerevisiae GAL1-GAL10 promoter was inserted upstream of the C. albicans HEX1 gene, which encodes N-acetylglucosaminidase. The gene was introduced into S. cerevisiae AH22, which has no background N-acetylglucosaminidase activity. Expression of HEX1 in transformed cells was induced significantly by galactose and was repressed by glucose. The HEX1 gene product was functional in S. cerevisiae cells and was targeted to the periplasm. Both untransformed S. cerevisiae cells and cells expressing HEX1 had similar growth curves and cell morphology indicating that expression of N-acetylglucosaminidase was not toxic to the host strain. These results demonstrate that the HEX1 gene can be a useful reporter of gene expression in S. cerevisiae.

Acetylglucosaminidase↗
PubMed1988

Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain.

Duchenne muscular dystrophy (DMD), a sex-linked degenerative disorder of the muscle, is one of the most common lethal genetic diseases in man. It affects about one male in 3,500, with an estimated one-third of cases being caused by new mutations. A less severe disease, Becker's muscular dystrophy (BMD), maps to the same chromosomal locus and is most probably an allelic form of DMD. Both diseases are sometimes associated with various degrees of mental retardation; the molecular basis of these phenotypes is unknown (for review, see ref. 1). The giant DMD gene spans approximately 2,000 kilobases (kb) (0.05% of the human genome) and encodes a 14-kb mRNA. The tissue-specificity of its expression has not been precisely determined. Monaco et al., using Northern blots, reported expression of the gene in human fetal skeletal muscle and small intestine but not in human fetal brain, or in human cultured myoblasts and transformed B and T cells. More recently, expression was detected in mouse skeletal and cardiac muscle, but not in mouse brain. Here we show, using a ribonuclease protection assay, that the DMD gene is developmentally regulated in rat and mouse myogenic cell cultures, and that it is expressed in rat and mouse striated muscle, in mouse smooth muscle and in rat, mouse and rabbit brain. We could not detect transcripts in other non-muscle tissues.

Amino Acid Sequence↗
PubMed1995

Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila.

We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster. Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4. These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy. Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies. We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time.

Animals↗