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PubMed1986

A transient expression assay for tissue-specific gene expression of alcohol dehydrogenase in Drosophila.

The regulation of expression of the alcohol dehydrogenase gene of Drosophila was examined by injecting plasmids containing the gene directly into preblastoderm embryos and subsequently staining for alcohol dehydrogenase activity in somatic cells of larvae and adults. The alcohol dehydrogenase genes introduced in this manner were expressed normally in both adults and larvae; i.e., alcohol dehydrogenase activity was found exclusively in tissues where it would normally be expressed. Activity was found in some cells in more than 90% of all surviving third instar larvae, but not all cells which would normally express the enzyme were positive, presumably due to the random distribution of the injected DNA to the cells of the embryo. Regulated expression was not dependent on the vector used: tissue-specific expression was obtained from alcohol dehydrogenase genes inserted in the P-element vector, Carnegie-4; in pBR322; in pUC18; or in bacteriophage lambda. The bulk of the injected DNA was not integrated into the chromosome and appeared to persist throughout development as supercoiled and nicked circles. Using the procedure and in vitro mutagenesis, we were able to show that the alcohol dehydrogenase gene was expressed in a normal tissue-specific manner in larvae if there were 777 nucleotides of upstream information present.

Alcohol Dehydrogenase↗
PubMed1989

Transient expression of a chondroitin sulfate-related epitope during cartilage histomorphogenesis in the axial skeleton of fetal rats.

A monoclonal antibody (MC21C), raised in mouse in response to a mixture of bone proteins, was found to exhibit a unique reactivity toward native chondroitin sulfate chains. Indirect immunofluorescence and immunoperoxidase assays were performed on tissue sections at different stages of fetal rat development, in order to investigate the distribution of the MC21C epitope during cartilage morphogenesis and differentiation. This extracellular marker was present in the sclerotome and its distribution subsequently followed the segmentation pattern of the precartilaginous vertebral column. In addition, changes in the MC21C-immunostaining pattern strongly correlated with the initial growth of the vertebrae. In the axial skeleton (spinal column, basis cranii), the immunostaining by MC21C was maximum in precartilaginous condensations and then rapidly disappeared during the process of chondrification. Also, the perinotochordal matrix was intensely immunostained.

Alcian Blue↗
PubMed1989

Transient expression of a Ca2+-activated Cl- current during development of quail sensory neurons.

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.

Animals↗
PubMed1990

Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development.

Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid outgrowth, and in preventing the onset of chondrogenesis.

Animals↗
PubMed1991

Isolation of cDNAs partially encoding four Xenopus Wnt-1/int-1-related proteins and characterization of their transient expression during embryonic development.

To begin to study the functions of the Wnt-1/int-1 gene family during vertebrate development, we have isolated four Xenopus laevis cDNAs encoding the partial sequence of proteins homologous to Wnt-1/int-1. Xwnt-3, Xwnt-4, Xwnt-5A, and Xwnt-8 demonstrate between 35 and 50% amino acid identity with X. laevis Wnt-1/int-1 and most cysteine residues are conserved. Xwnt-4 and Xwnt-3 transcripts are detected only during the neurula through tadpole stages of development. Expression of Xwnt-8 is observable during gastrulation, declines during neurulation, and is undetectable by the tadpole stage of development. Xwnt-5A transcripts are most prevalent in RNA from oocytes and tadpoles, although low level expression is detected at all stages examined. The temporal changes in expression of these transcripts imply a unique role for each Xwnt during embryogenesis.

Amino Acid Sequence↗
PubMed1992

Transient expression of syndecan in mesenchymal cell aggregates of the embryonic kidney.

Induction of the embryonic kidney mesenchyme is followed by formation of cell aggregates which subsequently transform into epithelial tubules. Syndecan, which binds various matrix components and growth factors, is a candidate molecule to be involved in this process. We have analyzed the changes in the expression of syndecan during tubule morphogenesis by using in situ hybridization and slot-blot analysis. The expression pattern of syndecan was compared with the distribution of cell proliferation analyzed by immunohistochemistry. Furthermore, the expression of syndecan during formation of the pretubular aggregates was studied in hanging-drop cultures of experimentally induced mesenchymal cells. Syndecan mRNA was expressed in the metanephric mesenchyme prior to induction, was intensely present during formation of the pretubular cell aggregates, but was lost during maturation of the nephron. Slot-blot analyses of the kidney mesenchymes (11-day kidney) cultured in a transfilter situation with a heterotypic inductor tissue that triggers a complete tubulogenic program in the nephric mesenchyme during the first 24 hr suggested the presence of syndecan mRNA in the uninduced mesenchymes with no change during induction. Expression of mRNA was stimulated later (13-day kidney) followed by subsequent decrease. Immunoisolation of sulfate-labeled syndecan, however, revealed a marked stimulation in the induced kidney mesenchyme during the first 24-hr inductive period when the DNA level still remained constant. In hanging-drop cultures where either induced or uninduced mesenchymal cells were dissociated and reaggregated, syndecan was detected only in the induced and aggregating mesenchymal cells. Double-immunostaining demonstrated a close correlation between syndecan expression and cell proliferation analyzed by bromodeoxyuridine incorporation. Thus, it appears that syndecan expression in the mesenchyme is initially induced post-transcriptionally and later during differentiation at the mRNA level. Syndecan may have a dual function during early kidney morphogenesis; it may be involved in cell aggregation through its adhesive properties, and it may contribute to proliferation of the induced mesenchymal cells by binding growth factors.

Animals↗
PubMed1990

Transient expression of the transformed phenotype stimulated by 12-O-tetradecanoyl phorbol-13-acetate.

The objective of the present work was to determine whether a short-term perturbation of cells by 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment caused shape changes identical to those found in oncogenic transformation. A cell line derived from the rat respiratory tract epithelium, 1000 W, was used, in which shape changes had been identified previously as the cells underwent transformation during long-term growth in vitro. These changes corresponded to a steeper rise of the cell from the substrate and a smoothing of the surface contours throughout the periphery of the cell. The phenotype was measured by maximum likelihood estimation, based on the values of several geometrical shape descriptors. With continuous TPA treatment, the cells adopted a transformed phenotype by 2 h. The effect was maximal by 5 h but began to decline by 10 h. Shape change in the opposite direction was stimulated by treatment with the protein kinase C inhibitor staurosporine, and its effects were counteracted if the cells were simultaneously exposed to TPA. No appreciable metabolism of [20-3H]TPA occurred until 24 h after treatment. Enumerating the shape descriptors whose values composed the transformed phenotype indicated that the TPA-stimulated changes were qualitatively similar to those accompanying oncogenic transformation. The subsequent alterations, however, involved few of the variables that composed the transformed phenotype and therefore did not represent a true reversal of the change. Changes observed up to 5 h were not dependent on new RNA synthesis but required continued protein synthesis.

Animals↗
PubMed1992

Mesostriatal dopaminergic axons transiently express high levels of NILE during development.

The distribution of nerve growth factor-inducible large external (NILE) glycoprotein in the developing mesostriatal dopaminergic system was studied by immunolabeling prenatal, postnatal, and adult rat brains for both NILE and tyrosine hydroxylase (TH). NILE-immunoreactive (NILE-IR) material outlined the TH-immunoreactive (TH-IR) neurons in the ventral mesencephalon at ages E14, E15, and E16 but not at later ages. Fascicles of TH-IR axons in the developing mesostriatal dopaminergic tract were strongly immunoreactive for NILE from E15 through E20. In the postnatal brain, NILE-IR material no longer clearly outlined the fascicles of dopaminergic axons as they ran from the mesencephalon to the striatum. In the striatum of postnatal and adult rats, NILE-IR material was diffusely distributed through the neuropil. Our observations suggest that NILE may play a role in guidance of growing dopaminergic axons in the developing mesostriatal tract and aggregation of the dopaminergic axons into fascicles.

Animals↗
PubMed1989

Sta!le and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways.

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Delta zero-thalassemia in cis of beta Knossos-globin gene. Normal structure transient expression of the delta-globin gene.

We have previously described the first homozygous cases of Hb Knossos in an Algerian family. The Hb A2 was completely absent, ascertaining the presence of a delta zero-thalassemia determinant in cis of the beta Knossos S gene. Here, we investigate the affected delta-globin gene. The complete DNA sequence of the gene and its 5' and 3' flanking regions was determined. Only two nucleotide changes were recorded: a C----T substitution at -199 and an AT insertion at -448 upstream from the cap site. To examine the involvement of these changes in gene function, the delta-gene was subcloned in an expression vector and introduced into COS cells. Analysis of RNA derived from these cells, using an S1 protection assay and dot-blot hybridization, revealed qualitatively and quantitatively normal transcription. The loss of delta-globin gene activity in vivo may be due to the alteration of a tissue-specific control.

DNA↗
PubMed1990

Interleukin-1 induces rapid and transient expression of the c-fos proto-oncogene in isolated pancreatic islets and in purified beta-cells.

The effect of interleukin-1 beta (IL-1) on expression of c-fos mRNA in isolated rat pancreatic islets was examined. Accumulation of c-fos mRNA was demonstrable after 30 min of exposure to IL-1, peaked by 60 min, and declined thereafter. Fluorescence-activated cell sorting (FACS) of dispersed islet cells was employed to localize the accumulation of c-fos mRNA to the beta-cell. Cycloheximide did not influence the induction of c fos mRNA by IL-1. Accumulation of c-fos mRNA therefore appears to be an early signal transduction event in the beta-cell and a component of the cellular mechanism(s) by which IL-1 influences beta-cell function.

Animals↗
PubMed1989

Transient expression of B19 parvovirus gene products in COS-7 cells transfected with B19-SV40 hybrid vectors.

Hybrid B19 parvovirus-SV40 origin vectors were transfected into COS-7 cells and replication of these plasmids studied. Plasmids that have a frameshift mutation within the nonstructural gene region replicated to high level (copy number approximately 10,000/transfected cell) although somewhat lower than pSVOd, the SV40 origin vector without B19 sequence (copy number approximately 100,000/transfected cell). However, hybrid B19 parvovirus-SV40 origin vectors that do not contain these frameshift mutations replicated to a much lower level (copy number approximately 1000/transfected cell). Although the hybrid vectors studied replicated at different efficiencies in COS-7 cells, they are transcribed at approximately the same level, resulting in RNA species that are indistinguishable from those seen in B19 virus-infected erythroid bone marrow cells. Western blot analysis demonstrated that the mRNAs are translated into polypeptides of the same size and, in the case of viral structural proteins, in same relative abundance as seen in a B19-infected clinical sample.

Blotting, Northern↗
PubMed1986

Transient expression of a 'lens-specific' gene, delta-crystallin, in the embryonic chicken adenohypophysis.

A characteristic protein of the lens, delta-crystallin, has been reported previously to be present in the embryonic chicken adenohypophysis. We confirmed this earlier finding by biochemical detection of delta-crystallin protein using a monoclonal antibody and delta-crystallin mRNA using a specific cDNA probe. We estimate the concentration of delta-crystallin and its mRNA in the 3.5-day embryonic chicken adenohypophysis to be approximately 1/3,000 and 1/5,000 of the respective value found in lens. Tissue culture revealed that cells positive for delta-crystallin comprise about 30% of embryonic adenohypophysis and are randomly scattered in this organ. No lentoid formation was observed during the culture period.

Animals↗
PubMed1991

Transient expression of trisomy 21 and monosomy 7 following cyclosporin A in a patient with aplastic anemia.

We present a patient with severe idiopathic aplastic anemia with no previous chromosomal abnormalities who developed trisomy 21 and monosomy 7 during treatment with intravenous (i.v.) cyclosporine. The abnormal karyotype disappeared when the drug was changed to the oral form. This cytogenetic aberration, previously unreported in association with cyclosporine, may reflect either a direct drug effect or the emergence of a hidden myelodysplastic cell clone subject to preferential survival during immunosuppression.

Administration, Oral↗
PubMed1985

Transient expression of tyrosine hydroxylase immunoreactivity in some neurons of the rat neocortex during postnatal development.

Tyrosine hydroxylase-like immunoreactive neurons were observed in the dorsolateral and medial neocortex of the rat during postnatal development. They occurred from 8 up to 24 days of age and lacked other catecholamine synthetizing enzymes. They appeared to be insensitive to the suppression of cortical noradrenergic innervation induced by neonatal subcutaneous injections of 6-hydroxydopamine.

Animals↗