Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.     

Comparison of messenger RNA in photoperiodically induced and noninduced Xanthium buds

In response to the floral stimulus, Xanthium buds synthesize relatively more messenger RNA than do vegetative buds. This is demonstrated by fractionation, on methylated albumin-kieselguhr columns, of a mixture of nucleic acids from vegetative and induced buds, one being labeled with uridine-H(3) and the other with uridine-2-C(14). While floral induction stimulates a small increase in messenger RNA synthesis as revealed by labeling intact plants, this difference can be magnified by labeling excised buds in solution. From experiments with excised buds from Xanthium plants, it is concluded that buds from photoperiodically induced plants contain more messenger RNA than buds from noninduced ones do.     

Ribosomes: effect of interferon on their interaction with rapidly labeled cellular and viral RNA's

Rapidly labeled RNA of mouse L cells and labeled RNA of Mengo virus, unlike cellular RNA labeled under steady-state conditions, form detectable complexes with L-cell ribosomes. These ribosome-RNA complexes formed in vitro appear analogous to those assembled during polysome formation in vivo. When ribosomes are prepared from L cells exposed to homologous interferon, their capacity to associate with cell messenger is preserved, while their ability to interact with viral RNA is markedly reduced. The ribosomes from cells exposed to interferon are thus altered selectively to permit only certain messages to be bound and translated.     

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.     

D-alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor

A D-alanine-containing peptide termed dermorphin, with potent opiate-like activity, has been isolated from skin of the frog Phyllomedusa sauvagei. Complementary DNA (cDNA) libraries were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of an L-amino acid to its D-isomer.     

Thiamine release from nerve membranes by tetrodotoxin

Tetrodotoxin (3 x 10(-8) molar) promoted the release of thiamine from perfused rat and frog nerve preparations in a manner similar to other neuroactive drugs. When the rats were injected with thiamine labeled with sulfur-35, analyses of brain, spinal cord, and sciatic nerve homogenates revealed labeled thiamine in membrane, synaptosomes, and mitochondrial subfractions. However, on incubation of these fractions with tetrodotoxin, thiamine was released only from the membrane fragments.     

Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation

Antisera to a synthetic c-myc peptide and to c-myc antigens synthesized from various portions of the human gene expressed in Escherichia coli were used in order to characterize the protein product of the human c-myc oncogene. Although the deduced molecular weight of the human c-myc protein is 49,000, these antisera precipitate a protein from human cells that migrates in sodium dodecyl sulfate-polyacrylamide gel as if its molecular weight were 65,000. In addition, the mouse c-myc protein, whether synthesized in cells or in a cell-free system directed by pure, synthetic messenger RNA, has analogous properties and is immunoprecipitated by the antiserum to the human c-myc protein. Similar proteins are immunoprecipitated from monkey, rat, hamster, and frog cells, suggesting evolutionary conservation of antigenic structure of the c-myc protein among vertebrates. In addition, and in a manner consistent with the behavior of its messenger RNA, the immunoprecipitable c-myc protein is sharply induced by the action of mitogens on resting human T cells.     

Messenger RNA in early sea-urchin embryos: size classes

Rapidly labeled RNA from four-cell embryos and blastulae of sea urchins was analyzed by sedimentation and for ability to form DNA-RNA hybrids. The RNA was derived from polyribosomes and from the "gel interphase," an extraction compartment resulting from treatment of whole embryos with phenol and known to be enriched with nuclei. The RNA from both sources displayed a high degree of structural complementarity to DNA. This DNA-like RNA of the polyribosomes sedimented in discrete classes, rather than in the sedimentation continuum demonstrable for the labeled RNA of the gel interphase. Thus messenger RNA appears to emerge in the cytoplasm in discrete size classes.     

Ribosomal RNA synthesis and processing in a particulate site in the HeLa cell nucleus

A particulate fraction has been isolated from detergent-prepared HeLa cell nuclei. The fraction consists largely of organelles that resemble the nucleoli of intact cells. The 45S RNA that is precursor to 28S and 18S ribosomal RNA is associated with the fraction. The 32S RNA that is labeled after the 45S RNA and is the apparent precursor to 28S RNA is also associated with the fraction. The nucleoplasm contains 28S RNA that behaves as an intermediate between the 32S nucleolar RNA and the 28S cytoplasmic RNA.     

Template activity of RNA from antibody-producing tissues

An RNA fraction, which represents a small percentage of cellular RNA and which has the characteristics of nuclear messenger RNA, has been isolated from the spleen and lymph nodes of immunized rats by successive phenol extractions of these tissues at increasing temperatures. This fraction increased the amount of protein synthesized in a cell-free extract of Escherichia coli as much as 35 times and directed the synthesis of proteins different from those of E. coli.     

Complementary RNA in nucleus and cytoplasm of mouse liver cells

The rapidly labeled RNA from both the nuclei and cytoplasm of mouse liver cells can be bound specifically to mouse DNA. The bound fraction differs in base composition and metabolic stability from the bulk RNA. There is considerable cross reaction between this RNA and the DNA obtained from calf thymus.     

Progesterone-dependent messenger RNA: heterospecific activity in vivo

The action of progesterone in mediating the synthesis of avidin by the chick oviduct can be simulated by the intraoviductal instillation of nitrocellulose-trapped RNA from hormonally prepared chick or pigeon oviduct. Similarly, the pigeon oviduct synthesizes avidin in response to chick oviduct RNA. Thus, a heterospecific transfer of hormonal stimulation, through the transfer of progesterone-induced RNA, is demonstrated. The biological activity is lost after digestion by pancreatic ribonuclease. The 50-fold purification achieved by nitrocellulose chromatography of the total RNA preparation suggests that the activity resides in a messenger RNA fraction.     

Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease

The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.     

Induction of mesoderm by a viral oncogene in early Xenopus embryos

During frog embryogenesis, mesoderm is specified in the equatorial region of the early embryo by a signal from the vegetal hemisphere. Prospective ectodermal cells dissected from the animal hemisphere can be respecified to form mesodermal tissues by recombination with vegetal tissue or by treatment with any of several polypeptide growth factors or growth factor-like molecules. Together with the discovery that several developmental mutations in Drosophila are in genes with significant homology to mammalian mitogens and oncogenes, these observations suggest that early developmental signals may use similar transduction pathways to mitogenic signals characterized in cultured mammalian cells. Whether mesoderm can be induced by activation of intracellular signal transduction pathways implicated in mitogenesis and oncogenesis has been investigated with the viral oncogene polyoma middle T. Microinjection of middle T messenger RNA into early embryos results in the respecification of isolated prospective ectodermal tissue to form characteristic mesodermal structures. Middle T in frog blastomeres appears to associate with cellular activities similar to those observed in polyoma-transformed mouse cells, and transformation-defective middle T mutants fail to induce mesoderm. These results suggest that early inductive signals and mitogenic and oncogenic stimuli may share common signal transduction pathways.     

Multipotent precursors can give rise to all major cell types of the frog retina

A prospective lineage analysis was performed to determine the variety of cell types that could be formed by individual precursor cells of the developing frog retina. Fluorescent dextran was iontophoretically injected into single cells of the embryonic optic vesicle. After further development of the embryo, labeled descendants were observed in all three layers of the larval retina. Furthermore, different clones were composed of various combinations of all major cell types, including the glial Müller cells. Hence, single optic vesicle cells have the potential to form any type of retinal cell, suggesting that the interactions that specify the differentiation pathway of retinal cells must occur late in development.     

Membranous structures associated with translation and transcription of poliovirus RNA

Poliovirus RNA and proteins are synthesized in association with distinct membranous structures that were separated by means of Isopycnic centrifugation of cytoplasmic extracts in discontinuous sucrose-density gradients. Viral RNA is replicated in a structure that contains rapidly labeled replicative intermediate RNA and viral RNA polymerase associated with the smooth membrane fraction. In sucrose gradients this viral RNA replication complex is distributed at densities in the range of 1.12 to 1.18 grams per cubic centimeter. Viral proteins are synthesized on polyribosomes bound to membranes and sediment with polyribosomes at densities of less than 1.25 grams per cubic centimeter.     

Rat brain N-methyl-D-aspartate receptors expressed in Xenopus oocytes

N-methyl-D-aspartate (NMDA) activates a class of excitatory amino acid receptor involved in a variety of plastic and pathological processes in the brain. Quantitative study of the NMDA receptor has been difficult in mammalian neurons, because it usually exists with other excitatory amino acid receptors of overlapping pharmacological specificities. Xenopus oocytes injected with messenger RNA isolated from primary cultures of rat brain have now been used to study NMDA receptors. The distinguishing properties of neuronal NMDA receptors have been reproduced in this amphibian cell, including voltage-dependent block by magnesium, block by the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid, and potentiation by glycine. This preparation should facilitate the quantitative study of the regulation of NMDA receptor activation and serve as a tool for purification of the encoding messenger RNA.     

Breakdown of rat-liver ergosomes in vivo after actinomycin inhibition of messenger RNA synthesis

Ribosomes isolated from rat liver occur predominantly in the form of aggregates (ergosomes) corresponding to multiples of 73S particles held together by messenger RNA. After injecting rats with actinomycin, these aggregates gradually break down in vivo to 73S monomers and 113S dimers. We conclude that the observed breakdown results from the degradation of messenger RNA and the prevention by actinomycin of the synthesis of new messenger RNA.     

Ectopic pro-opiolipomelanocortin: sequence of cDNA coding for beta-melanocyte-stimulating hormone and beta-endorphin

A recombinant bacterial plasmid, pMS1, was constructed that contains 318 nucleotides complementary to a portion of pro-opiolipomelanocortin (proOLMC) messenger RNA from an ectopic adrenocorticotropin-producing tumor. The cloned complementary DNA insert, which contains the sequence that codes for all of the beta-melanocyte-stimulating hormone and beta-endorphin portions of proOLMC, as well as the 3' nontranslated section, is identical to the genomic sequence. Hybridization of tumor proOLMC complementary DNA to RNA subjected to electrophoresis and transferred to a nitrocellulose filter revealed two proOLMC messenger RNA species in the tumor polyadenylated RNA, but only one in pituitary polyadenylated RNA. At least one of the tumor proOLMC messenger RNA's is similar, if not identical, to human pituitary proOLMC messenger RNA.