An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers.

The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.

Distinct spatial localization of specific mRNAs in cultured sympathetic neurons.

We examined the subcellular distribution of specific mRNAs in cultured sympathetic neurons. Under appropriate conditions, sympathetic neurons extend both axons and dendrites that are distinguishable by light microscopic and immunocytochemical criteria. In situ hybridization revealed a differential localization of mRNA within dendrites. mRNA encoding MAP2 was abundant in cell bodies and distributed nonhomogeneously throughout the dendritic compartment, but was not detected in axons. In contrast, mRNAs encoding GAP-43 and alpha-tubulin were restricted to the cell body and largely excluded from dendrites as well as axons. Detergent extraction revealed that most dendrite-associated mRNA encoding MAP2 was associated with the Triton X-100 insoluble fraction of the cell. The subset of mRNAs present in the dendritic compartment may encode proteins involved in the morphogenesis and remodeling of dendrites.

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites.

We have examined the morphology of fetal rat sympathetic neurons grown in serum-free medium in the absence of nonneuronal cells. Because cell density can affect phenotypic expression in vitro, the morphological analysis was subdivided into the study of isolated neurons (neurons whose somata were at least 150 micron from their nearest neighbor) and of more highly aggregated neurons. When isolated neurons were injected with intracellular markers, it was found that most (79%) had a single process emanating from their somata and that this unipolar state persisted for at least 8 weeks in vitro. The processes of unipolar sympathetic neurons had the appearance of axons in that they were thin and long, had a constant diameter, and were relatively unbranched. Cytochemical methods revealed that such processes had other axonal characteristics: (1) they were more reactive with a monoclonal antibody against phosphorylated forms of the M and H neurofilament subunits than with an antibody to nonphosphorylated forms of these proteins; (2) they also reacted with antibodies to the tau microtubule-associated protein and to the phosphorylated forms of the H neurofilament subunit; and (3) they contained only small amounts of RNA as determined by [3H]uridine autoradiography. These data indicate that neurons which normally form dendrites in vivo need not express this capacity in vitro and that axonal and dendritic growth can be dissociated under some conditions in culture. While most isolated neurons were unipolar, neurons in regions of high neuronal cell density were usually multipolar. In addition to axons, multipolar neurons had processes with some of the characteristics expected of rudimentary dendrites: they ended locally (usually within 100 micron), were often highly branched, and reacted with an antibody to nonphosphorylated forms of the M and H neurofilament subunits. The effects of density were most prominent when neurons were within aggregates in which the somata were in close apposition. Density-dependent changes in morphology were less frequently observed when neuronal somata were separated by greater distances (30-100 micron). These data indicate that the morphology of sympathetic neurons is subject to environmental regulation and that neuron-neuron interactions can promote the extension of rudimentary dendrites in vitro.

Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth.

In the preceding paper, we reported that embryonic rat sympathetic neurons formed axons, but not dendrites, when they were maintained in the absence of serum and nonneuronal cells. To assess the effects of serum-derived factors on cellular morphology, cultures were initially maintained in serum-free medium while nonneuronal cells were eliminated. Subsequently some cultures were chronically exposed either to fetal calf serum (10%) or to a high-molecular-weight ammonium sulfate fraction of serum (P40 material, 500 micrograms/ml). Phase-contrast microscopy revealed that serum and P40 material did not alter neuronal survival, but did cause flattening of the somata and fasciculation of processes. When neurons exposed to serum or P40 material were injected with Lucifer Yellow, it was found that the majority (greater than or equal to 90%) had local, tapered processes that could be identified as dendrites by light microscopic criteria. These local processes also exhibited other dendritic characteristics in that (1) they reacted with monoclonal antibodies to nonphosphorylated forms of the M and H neurofilament subunits and to microtubule-associated protein 2; and (2) they had substantial amounts of RNA as determined by [3H]uridine autoradiography. Quantitative measurements of the effects of serum and P40 material on dendritic morphology revealed that (1) an 8-day exposure caused most neurons (greater than 80%) to form dendrites; (2) neurons typically had more than one dendrite (mean of 4.1 +/- 0.2 dendrites/cell after a 28-day exposure); and (3) the dendrites were relatively short with the maximum extent of the dendritic arbor being 110 +/- 13 micron after 4 weeks. Serum and P40 material did not routinely cause the formation of supernumerary axons, did not alter radial axonal outgrowth from ganglion explants, and did not significantly increase [3H]leucine incorporation. Thus, serum contains a factor (or factors) which selectively stimulates the extension of dendrites, but not axons. If such a factor were operative in situ, it could play an important role in determining the morphology of sympathetic neurons. In examining the mechanism of serum-induced dendritic growth, we found that even high concentrations (5 micrograms/ml) of nerve growth factor failed to promote dendritic growth in the absence of serum; thus, nerve growth factor by itself is not a sufficient condition for the extension of dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Avian sarcoma virus gag precursor polypeptide is not processed in mammalian cells.

We studied intracellular avian gag proteins (internal structural proteins of virions) in several mammalian cell lines transformed by Rous sarcoma virus. All lines examined contain gag antigens as determined by radioimmune assay. We used the techniques of protein blotting from polyacrylamide gels, which detects nanogram quantities of viral protein, to investigate the size of intracellular viral polypeptides. All of the lines that contained enough viral protein to be amenable to this type of analysis synthesized Pr76, the avian sarcoma virus gag precursor polypeptide, but failed to process it into mature virion proteins. In some cell lines, the recovery of Pr76 was greatly enhanced by the addition of a mixture of protease inhibitors, including the sulfhydryl-blocking reagent N-ethylmaleimide, to the lysis buffer. At least several of the mammalian cells also synthesized a viral polypeptide the size of Pr180, the precursor to reverse transcriptase. Since Rous sarcoma virus does not replicate or replicates extremely poorly in mammalian cells, the lack of processing suggests that cleavage and virion assembly are invariably associated.