Mutant alleles of Arabidopsis RADIALLY SWOLLEN 4 and 7 reduce growth anisotropy without altering the transverse orientation of cortical microtubules or cellulose microfibrils

Development ◽  
2002 ◽  
Vol 129 (20) ◽  
pp. 4821-4830 ◽  
Author(s):  
Allison M. D. Wiedemeier ◽  
Jan E. Judy-March ◽  
Charles H. Hocart ◽  
Geoffrey O. Wasteneys ◽  
Richard E. Williamson ◽  
...  
Keyword(s):  
Mechanical Anisotropy ◽  
Cellulose Microfibrils ◽  
Analytical Determination ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Cortical Microtubules ◽  
Cell Production ◽  
Anisotropic Expansion ◽  
Anisotropic Mechanical Properties

The anisotropic growth of plant cells depends on cell walls having anisotropic mechanical properties, which are hypothesized to arise from aligned cellulose microfibrils. To test this hypothesis and to identify genes involved in controlling plant shape, we isolated mutants in Arabidopsis thaliana in which the degree of anisotropic expansion of the root is reduced. We report here the characterization of mutants at two new loci, RADIALLY SWOLLEN 4 (RSW4) and RSW7. The radial swelling phenotype is temperature sensitive, being moderate (rsw7) or negligible (rsw4) at the permissive temperature, 19°C, and pronounced at the restrictive temperature, 30°C. After transfer to 30°C, the primary root’s elongation rate decreases and diameter increases, with all tissues swelling radially. Swelling is accompanied by ectopic cell production but swelling is not reduced when the extra cell production is eliminated chemically. A double mutant was generated, whose roots swell constitutively and more than either parent. Based on analytical determination of acid-insoluble glucose, the amount of cellulose was normal in rsw4 and slightly elevated in rsw7. The orientation of cortical microtubules was examined with immunofluorescence in whole mounts and in semi-thin plastic sections, and the orientation of microfibrils was examined with field-emission scanning electron microscopy and quantitative polarized-light microscopy. In the swollen regions of both mutants, cortical microtubules and cellulose microfibrils are neither depleted nor disoriented. Thus, oriented microtubules and microfibrils themselves are insufficient to limit radial expansion; to build a wall with high mechanical anisotropy, additional factors are required, supplied in part by RSW4 and RSW7.

Auxin and Cell Wall Crosstalk as Revealed by the Arabidopsis thaliana Cellulose Synthase Mutant Radially Swollen 1

2019 ◽  
Vol 60 (7) ◽  
pp. 1487-1503 ◽  
Author(s):  
Thiel A. Lehman ◽  
Karen A Sanguinet
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Wall ◽  
Cellulose Synthase ◽  
Cellulose Microfibrils ◽  
Single Amino Acid ◽  
Cell Wall Biosynthesis ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Anisotropic Expansion ◽  
Single Amino Acid Change

AbstractPlant cells sheath themselves in a complex lattice of polysaccharides, proteins and enzymes forming an integral matrix known as the cell wall. Cellulose microfibrils, the primary component of cell walls, are synthesized at the plasma membrane by CELLULOSE SYNTHASE A (CESA) proteins throughout cellular growth and are responsible for turgor-driven anisotropic expansion. Associations between hormone signaling and cell wall biosynthesis have long been suggested, but recently direct links have been found revealing hormones play key regulatory roles in cellulose biosynthesis. The radially swollen 1 (rsw1) allele of Arabidopsis thaliana CESA1 harbors a single amino acid change that renders the protein unstable at high temperatures. We used the conditional nature of rsw1 to investigate how auxin contributes to isotropic growth. We found that exogenous auxin treatment reduces isotropic swelling in rsw1 roots at the restrictive temperature of 30�C. We also discovered decreases in auxin influx between rsw1 and wild-type roots via confocal imaging of AUX1-YFP, even at the permissive temperature of 19�C. Moreover, rsw1 displayed mis-expression of auxin-responsive and CESA genes. Additionally, we found altered auxin maxima in rsw1 mutant roots at the onset of swelling using DII-VENUS and DR5:vYFP auxin reporters. Overall, we conclude disrupted cell wall biosynthesis perturbs auxin transport leading to altered auxin homeostasis impacting both anisotropic and isotropic growth that affects overall root morphology.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

1986 ◽  
Vol 6 (12) ◽  
pp. 4594-4601
Author(s):  
J J Dermody ◽  
B E Wojcik ◽  
H Du ◽  
H L Ozer
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Viral Dna ◽  
Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6350-6360
Author(s):  
F Houman ◽  
C Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Essential Gene ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Nonsense Mutations ◽  
Carboxy Terminal

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes

1988 ◽  
Vol 255 (3) ◽  
pp. C261-C270 ◽  
Author(s):  
M. E. Handlogten ◽  
M. S. Kilberg
Keyword(s):  
Cell Density ◽  
De Novo ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
System A

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability

Brain ◽  
2019 ◽  
Vol 143 (1) ◽  
pp. 112-130 ◽  
Author(s):  
Nicole J Van Bergen ◽  
Yiran Guo ◽  
Noraldin Al-Deri ◽  
Zhanna Lipatova ◽  
Daniela Stanga ◽  
...  
Keyword(s):  
Neurological Disorders ◽  
Single Nucleotide Polymorphism Array ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cerebellar Atrophy ◽  
Sensorineural Deafness ◽  
Size Exclusion ◽  
Autophagic Flux ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature

Abstract The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

scholarly journals Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein

2004 ◽  
Vol 78 (8) ◽  
pp. 3880-3888 ◽  
Author(s):  
Ana M. Falcón ◽  
Rosa M. Marión ◽  
Thomas Zürcher ◽  
Paulino Gómez ◽  
Agustín Portela ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Ns1 Protein ◽  
Late Gene Expression ◽  
Infected Cells ◽  
Defective Rna ◽  
Nucleocytoplasmic Export

ABSTRACT Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.

scholarly journals Regulation of type VI collagen synthesis in transformed mesenchymal cells

1988 ◽  
Vol 253 (2) ◽  
pp. 381-386 ◽  
Author(s):  
T Schreier ◽  
R R Friis ◽  
K H Winterhalter ◽  
B Trüeb
Keyword(s):  
Viral Vectors ◽  
Collagen Synthesis ◽  
Inhibitory Effect ◽  
Mesenchymal Cells ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Type Vi Collagen ◽  
Rous Sarcoma ◽  
The Individual

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the ‘nuclear’ oncogene v-myc had an inhibitory effect similar to that of the ‘cytoplasmic’ oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.

scholarly journals Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants

2007 ◽  
Vol 81 (6) ◽  
pp. 2849-2860 ◽  
Author(s):  
Giuseppe Balistreri ◽  
Javier Caldentey ◽  
Leevi Kääriäinen ◽  
Tero Ahola
Keyword(s):  
Protease Activity ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Helicase Domain ◽  
Permissive Temperature ◽  
Protease Domain ◽  
Rna Triphosphatase ◽  
Interdomain Interactions

ABSTRACT We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24°C and 35°C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35°C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.

scholarly journals Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

1991 ◽  
Vol 113 (6) ◽  
pp. 1313-1330 ◽  
Author(s):  
R J Duronio ◽  
D A Rudnick ◽  
R L Johnson ◽  
D R Johnson ◽  
J I Gordon
Keyword(s):  
Myristic Acid ◽  
Fatty Acid Synthetase ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Myristoyl Coa

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.

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