Defective intercellular cohesion in glycosylation mutants of Dictyostelium discoideum

In order to identify the biological roles of protein-linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature-sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA-resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA-resistant cohesion, and reverted almost totally to wild-type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA-resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N-linked oligosaccharides in aggregation-stage intercellular cohesion.     

Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway

Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway. In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro. At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates. In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed. The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus. Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.     

Temperature-Sensitive, Assembly-Defective Flagella Mutants of CHLAMYDOMONAS REINHARDTII

We describe an efficient selection procedure for the isolation of mutants of Chlamydomonas reinhardtii with temperature sensitive flagella defects, with final yields of up to 11% of the population being mutant. Several mutants, all showing an inability to maintain flagellar integrity at the restrictive temperature, are described. We have examined flagellar stability and reassembly at various temperatures in the mutants. Mapping data are provided for these, as well as for some previously described mutants.     

Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants

Utilization of temperature-sensitive mutants of Tetrahymena pyriformis affected in cell division or developmental pathway selection has permitted elucidation of causal dependencies interrelating micronuclear and macronuclear replication and division, oral development, and cytokinesis. In those mutants in which cell division is specifically blocked at restrictive temperatures, micronuclear division proceeds with somewhat accelerated periodicity but maintains normal coupling to predivision oral development. Macronuclear division is almost totally suppressed in an early acting mutant (mola) that prevents formation of the fission zone, and is variably affected in other mutants (such as mo3) that allow the fission zone to form but arrest constriction. However, macronuclear DNA synthesis can proceed for about four cycles in the nondividing mutant cells. A second class of mutants (psm) undergoes a switch of developmental pathway such that cells fail to enter division but instead repeatedly carry out an unusual type of oral replacement while growing in nutrient medium at the restrictive temperature. Under these circumstances no nuclei divide, yet macronuclear DNA accumulation continues. These results suggest that (a) macronuclear division is stringently affected by restriction of cell division, (b) micronuclear division and replication can continue in cells that are undergoing the type of oral development that is characteristic of division cycles, and (c) macronuclear DNA synthesis can continue in growing cells regardless of their developmental status. The observed relationships among events are consistent with the further suggestion that the cell cycle in this organism may consist of separate clusters of events. with a varying degree of coupling among clusters. A minimal model of the Tetrahymena cell cycle that takes these phenomena into account is suggested.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

S-phase, G2, and nuclear division mutants of Aspergillus nidulans

Twenty-two temperature-sensitive cell cycle mutants of the fungus Aspergillus nidulans, which block in interphase at restrictive temperature, were analyzed by the reciprocal shift method of Jarvik and Botstein (Proc. Nath Acad. Sci. U.S.A. 70:2046-2050, 1973) and Hereford and Hartwell (J. Mol. Biol. 84:445-461, 1974) to determine whether these mutations were blocked at the G1, S, or G2 phase of the cell cycle. We found five mutants to be blocked in S and nine to be blocked in G2. Two of the G2 mutants were atypical in that they were not able to accomplish the G2 to M transition at restrictive temperature but nevertheless could initiate subsequent cycles of DNA replication. None was blocked in G1. There were nine strains that could not be classified. The block imposed by restrictive temperature was irreversible in three of these strains, and the six other strains were unclassifiable due to their aberrant terminal nuclear phenotypes.     

Conditional intercellular cohesion in a Dictyostelium discoideum mutant which is temperature sensitive for correct processing of asparagine-linked oligosaccharides.

Mutants of Dictyostelium discoideum have been isolated by a selection for cells with temperature-sensitive defects in the maturation of glycoprotein N-linked oligosaccharides. Here we describe a mutant, HT7, which is unable to aggregate at the restrictive temperature, but which aggregates and makes fruiting bodies at the permissive temperature. HT7 shows normal early developmental intercellular cohesion, but is temperature sensitive for expression of the ethylenediamine-tetraacetic acid (EDTA)-resistant cohesion characteristic of aggregation. The mutant initiates aggregation, but forms only loose cell mounds which later disperse. Metabolic labelling studies indicate that the thermolabile defect is not in protein synthesis, assembly of the lipid-linked precursor of N-linked oligosaccharides or transfer of the precursor to proteins. However, the defect does prevent assembly of fully processed N-linked oligosaccharides. Further, two glycopeptides, obtained from exhaustive Pronase digests of wild-type plasma membrane glycoproteins, inhibit intercellular cohesion of aggregation-stage wild-type cells. HT7 produces only approximately 50% of the wild-type level of these glycopeptides at the restrictive temperature and one of the glycopeptides has reduced cohesion inhibition ability. A revertant of HT7 was found to aggregate normally, to have restored EDTA-resistant cohesion, to have normal profiles of N-linked oligosaccharides and to express the two cohesion-inhibiting glycopeptides normally. These data strongly support a model in which cohesion during late aggregation is at least in part due to recognition between surface glycans and receptors on neighbouring cells.     

Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection

We have adapted a [3H]mannose suicide selection to identify mutations in additional genes which function in the early part of the yeast secretory pathway. Thus far this protocol has led to the identification of two new genes which are implicated in this process, as well as additional alleles of previously identified genes. The new mutants, bet1 and bet2, are temperature sensitive for growth and protein transport. Thin section analysis has revealed the accumulation of a network of endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). Precursors of exported proteins that accumulate in the cell at 37 degrees C are terminally core glycosylated. These observations suggest that the transport of precursors is blocked subsequent to translocation into the ER but before entry into the Golgi apparatus. The bet1 and bet2 mutants define two new complementation groups which have the same properties as previously identified ER-accumulating mutants. This and previous findings (Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:205-215) suggest that protein exit from the ER and entry into the Golgi apparatus is a complex process requiring at least 11 genes.     

Secretion of Saccharomyces cerevisiae Killer Toxin: Processing of the Glycosylated Precursor

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2. Protoxin stability in kex1 kex2 double mutants indicated the order kex1 --> kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1- and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.     

Mutations affecting antigen processing impair class II-restricted allorecognition

Both exogenously derived and endogenously derived Ag generally require processing for their optimal binding and presentation by class I and class II major histocompatibility proteins. It is not known whether steps involved in Ag processing also affect the recognition of alloreactive T cells. We have recently described B cell mutants which have general defects in the processing and presentation of a variety of exogenous Ag to class II restricted T cells. In this report we have studied the ability of these processing mutants to stimulate a set of anti-DR3-specific alloreactive T cells clones. These processing/presentation mutants express normal MHC class II molecules, both in terms of primary sequence and cell surface abundance, but they appear unable to generate effective peptide-MHC complexes. When tested for their ability to stimulate MHC class II alloreactive T cell clones, only one of four T cell clones was stimulated by these mutants; the other three alloreactive T cell clones were not stimulated by either of two different mutants. Both of these mutants express normal levels of the accessory molecules, LFA-3 and ICAM-1. The inability of these mutants to stimulate three of four alloreactive clones indicates that the capacity to be recognized by many alloreactive T cells is linked to the Ag processing capacity of a stimulator cell.     

The Escherichia coli dnaJ mutation affects biosynthesis of specific proteins, including those of the lac operon.

Temperature-sensitive dnaJ mutants of Escherichia coli showed a thermosensitive defect in the synthesis of beta-galactosidase. Synthesis of the lac mRNA was greatly reduced at the restrictive temperature. The mutants were also conditionally defective in the synthesis of a subset of membrane proteins such as succinate dehydrogenase, whereas the synthesis of anthranilate synthetase, encoded by trpED, as well as that of most cellular proteins, was unaffected at the restrictive temperature. The defect was specific for the dnaJ mutants among several dna mutants which are known to be involved in the initiation of DNA synthesis: dnaK, dnaA, and dnaB mutants synthesized each of these proteins normally even at the restrictive temperature. At the restrictive temperature, growth of the dnaJ mutants was arrested at a specific stage of the cell cycle.     

Viral DNA Synthesis in Cells Infected by Temperature-Sensitive Mutants of Simian Virus 40

Temperature-sensitive mutants of simian virus 40 (SV40) have been classified as those that are blocked prior to viral DNA synthesis at the restrictive temperature, "early" mutants, and those harboring a defect later in the replication cycle, "late" mutants. Mutants of the A and D complementation groups are early, those of the B, C, and BC groups are late. Our results confirm earlier reports that A mutants are defective in a function required for the initiation of each round of viral DNA synthesis. D mutants, on the other hand, continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature. The length of time required for D function to be expressed at the permissive temperature-after which infection proceeds unabated on shifting of the cultures to the restrictive temperature-is 10 to 20 h. The viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.     

shibire, a neurogenic mutant of Drosophila

Embryos of the temperature-sensitive mutant shibirets 1 were given short exposures to the restrictive temperature during the stage when neuroblasts segregate from the presumptive epidermis. The resulting lethal phenotype, expansion of the nervous system at the expense of the epidermis, is characteristic of a group of mutants called neurogenic mutants. Exposures as short as 20 min were sufficient to promote the neurogenic phenotype. Cell masses from heat-pulsed embryos could be cultured in vivo as tumorous masses which retained some characteristics of neural tissue. An examination of the neurogenic region from heat-pulsed embryos revealed numerous packets of extracellular vesicles and coated pits blocked in endocytosis.     

Behavioral and Biochemical Defects in Temperature-Sensitive Acetylcholinesterase Mutants of DROSOPHILA MELANOGASTER

Temperature-sensitive (ts) mutants of the Ace gene, which codes for acetylcholinesterase (AChE) in Drosophila melanogaster, were analyzed for defects in viability, behavior and function of the enzyme. The use of heat-sensitive and cold-sensitive mutations permited the function of AChE in the nervous system to be analyzed temporally. All ts mutations were lethal, or nearly so, when animals expressing them were subjected to restrictive temperatures during late embryonic and very early larval stages. Heat treatments to Ace-ts mid- and late larvae had little effect on the behavior of these animals or on the viability or behavior of the eventual adults. Heat-sensitive mutants exposed to nonpermissive temperatures as pupae, by contrast, had severe defects in phototaxis and locomotor activity as adults. AChE extracted from adult ts mutants that had developed at a permissive temperature were abnormally heat labile, and they had reduced substrate affinity when assayed at restrictive temperatures. However, enzyme activity did not decline during exposure of heat-sensitive adults to high temperatures even though such treatments caused decrements in phototaxis (29°) and, eventually, cessation of movement (31°). The cold-sensitive mutant also produced readily detectable levels of AChE when exposed to a restrictive temperature during the early developmental stage when this mutation causes almost complete lethality. We suggest that the relationship among the genetic, biochemical and neurobiological defects in these mutants may involve more than merely temperature-sensitive catalytic functions.     

Temperature-Sensitive Mutants of a Chinese Hamster Cell Line. I. Selection of Clones with Defective Macromolecular Biosynthesis

Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxyuridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39degrees and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39degrees to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115-47 and 115-53) also stop incorporating radioactive amino acids and uridine within six hours at 39degrees. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33 degrees after 8 hours at 39 degrees, both resumed DNA synthesis immediately (less than 1 hour). Reversal of defective DNA synthesis in both mutants were sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication.     

DNA supercoiling in gyrase mutants.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.     

Functional Alcohol Dehydrogenase Mutants of Saccharomyces cerevisiae Conferring Temperature-Conditional Allyl Alcohol Resistance

Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI. Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered. The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible. Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance. Haploid cells of one of these mutants grow well on plates at 10 mM allyl alcohol at 19 degrees, but not at 37 degrees, the restrictive temperature. A second mutant grows well at 10 mM at 37 degrees, but its growth is restricted at 19 degrees. What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature. This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.