Enveloped viruses are excellent tools for the study of the biogenesis of epithelial polarity, because they bud asymmetrically from confluent monolayers of epithelial cells and because polarized budding is preceded by the accumulation of envelope proteins exclusively in the plasma membrane regions from which the viruses bud. In this work, three different experimental approaches showed that the carbohydrate moieties do not determine the final surface localization of either influenza (WSN strain) or vesicular stomatitis virus (VSV) envelope proteins in infected Madin-Darby Canine Kidney (MDCK) cells, as determined by immunofluorescence and immunoelectron microscopy, using ferritin as a marker. Infected concanavalin A- and ricin 1-resistant mutants of MDCK cells, with alterations in glycosylation, exhibited surface distributions of viral glycoproteins identical to those of the parental cell line, i.e., influenza envelope proteins were exclusively found in the apical surface, whereas VSV G protein was localized only in the basolateral region. MDCK cells treated with tunicamycin, which abolishes the glycosylation of viral glycoproteins, exhibited the same distribution of envelope proteins as control cells, after infection with VSF or influenza. A temperature-sensitive mutant of influenza WSN, ts3, which, when grown at the nonpermissive temperature of 39.5 degrees C, retains the sialic acid residues in the envelope glycoproteins, showed, at both 32 degrees C (permissive temperature) and 39.5 degrees C, budding polarity and viral glycoprotein distribution identical to those of the parental WSN strain, when grown in MDCK cells. These results demonstrate that carbohydrate moieties are not components of the addressing signals that determine the polarized distribution of viral envelope proteins, and possibly of the intrinsic cellular plasma membrane proteins, in the surface of epithelial cells.

A temperature-sensitive growth mutant derived from the BHK 21 cell line, ts AF8, was found to have greatly reduced DNA synthesis at the nonpermissive temperature. This reduction is mainly due to a decrease in the frequency of cells synthesizing DNA. Upon shift up, ts AF8 becomes blocked in the G1 phase of the cell cycle. The cells acquire elevated CAMP levels and a unimodal distribution of DNA content, equivalent to that of G1 cells at the permissive temperature. Ts AF8 cells blocked at the Gl/S boundary with hydroxyurea will enter S when shifted to the nonpermissive temperature. On the other hand, ts AF8 cells arrested in G1 by serum deprivation and shifted to the nonpermissive temperature at the moment of serum addition do not enter S, while those synchronized by isoleucine deprivation and shifted at the time of isoleucine addition will enter S . These data suggest that the cycle arrest point of the ts AF8 mutation is located in G1 between the blocks induced by serum starvation and isoleucine deprivation. The reduction in DNA synthesis caused by the ts AF8 mutation is not reversed by infection or transformation with Polyoma virus. Mitochondria1 DNA continues to be synthesized at wild-type levels at the nonpermissive temperature.

Two lectin-resistant mutants derived from Madin Darby canine kidney cells, with constitutive alterations in the asparagine-linked carbohydrate moieties, retained the characteristic structural and functional epithelial polarity of the parental cells. A ricin-resistant cell line was unable to incorporate galactose-sialic acid into glycoproteins and, from the pattern of cross-resistance to other lectins, appears to be different from previously described lines resistant to this lectin; the mutation in a concanavalin A-resistant line results, probably, in the production of defective carbohydrate cores of glycoproteins. In spite of glycosylation defects which result in an increased electrophoretic mobility of many cellular glycoproteins, both mutants retained the typical asymmetric structure of the plasma membrane (microvilli on the apical surface, junctional elements on the basolateral surface), functional tight junctions, and unidirectional active transport of electrolytes and water. These results suggest that glycoproteins with terminal galactosesialic acid moieties are not critically involved in the development and maintenance of polarity in epithelial cells. The mutant cells, particularly the ricin-resistant line, exhibited, however, morphological and electrophysiological changes which suggest a quantitative effect of the mutations on intracellular traffic of membranes and tight junction formation. The cell lines described in this paper, the first lectinresistant mutants of epithelial lineage, should prove useful tools for studying the peculiarities of glycosylating pathways in polarized cells.

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We have characterized a temperature-sensitive (ts) mutant of the hamster cell line BHK 21 that appears to have a defect in the processing of ribosomal RNA precursors at 39°. Mutant ts 422E grows at a normal rate at 330, but upon shift to 39°growth stops after about one cell doubling. The appearance of 28S rRNA and large ribosomal subunits in the cytoplasm of ts 422E at 390 is inhibited by about 95%, when compared to wild-type BHK cells. Production of 18S rRNA and small ribosomal subunits is unaffected. Shift-up experiments show that the defect in 28S rRNA production can be detected as early as 2-3 hr after the shift to 39°. Synthesis of the larger rRNA precursor is normal at high temperature, but the processing appears to be arrested after the formation of 32S rRNA. 32S rRNA accumulates to some extent in the nucleoli of ts 422E. ts 422E cells appear to have a single mutation, directly affecting the conversion of 32S to 28S rRNA. The reduced amount of 28S rRNA in the cytoplasm of ts 422E cells at 390 seems therefore responsible for their inability to grow at this temperature.In a previous publication, we described the isolation and the general properties of several temperature-sensitive (ts) mutants of the Syrian hamster cell line BHK 21/13 (1). We report now on the characterization of one of these mutants, ts 422E, whose inability to multiply at 390 seems to be due to a ts defect in the production of 28S ribosomal RNA (rRNA).Ribosomal RNA in eukaryotic cells is synthesized as a large precursor molecule that contains the sequences of one molecule of 28S RNA (the RNA component of the large ribosomal subunit) and of one molecule of 18S RNA (the RNA component of the small ribosomal subunit), and a nonribosomal portion, destined to be discarded (2, 3). As determined mostly from studies with HeLa cells, 45S RNA precursor molecules undergo chemical modifications through the activity of methylating enzymes. Processing then occurs in a stepwise fashion within the nucleolus, resulting in the appearance of RNA intermediates and formation of ribosomal subunits that are transferred to the cytoplasm [for a review see (4) ].In the ts BHK mutant 422E, rRNA production is fully normal at low temperature, but at 390 the appearance of 28S rRNA in the cytoplasm is greatly inhibited. The mechanism responsible for this defect seems to reside in an abnormal processing of rRNA precursors arrested at the 32S stage. MATERIALS AND METHODSCells. Cell Fractionation. Cells were fractionated into nuclear and cytoplasmic fractions essentially as described by Penman (5, 6). Cells were detached from the plate with a purified trypsin-EDTA solution, washed twice by centrifugation with cold isotonic Tris-buffered saline (pH 7.4), resuspended in hypotonic buffer (10 mM NaCl-1.5 mM MgCl2-10 mM Tris -HC1, pH 7.2) at a density of about 107 cells per ml, and allowed to swell for 10 min at 00. They were brokenwith a Dounce homogenizer and Nonidet P40 was added to a concentration of 1%.In some experiments, in order to obtain more purified nuclei a ...