scholarly journals The Dominant Temperature-Sensitive Lethal DTS7 of Drosophila melanogaster Encodes an Altered 20S Proteasome β-Type Subunit

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 211-220 ◽  
Author(s):  
Kerrie A Smyth ◽  
John M Belote
Keyword(s):  
Missense Mutation ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
20S Proteasome ◽  
Dependent Manner ◽  
Subunit Gene ◽  
Ambient Temperatures ◽  
Similar Phenotype ◽  
Allele Specific ◽  
Dominant Temperature Sensitive

Abstract Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 α-type and 7 β-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29° that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a β-type subunit gene, β2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another β-type subunit gene, β6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as “poison subunits,” disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.

scholarly journals The generation and genetic analysis of suppressors of lethal mutations in the Caenorhabditis elegans rol-3(V) gene.

Genetics ◽  
1994 ◽  
Vol 136 (1) ◽  
pp. 129-143 ◽  
Author(s):  
W B Barbazuk ◽  
R C Johnsen ◽  
D L Baillie
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Analysis ◽  
Genetic Interaction ◽  
Temperature Sensitive ◽  
Left Hand ◽  
Larval Stages ◽  
Allele Specific ◽  
Complementation Groups ◽  
Nematode Cuticle ◽  
Developmental Role

Abstract The Caenorhabditis elegans rol-3(e754) mutation is a member of a general class of mutations affecting gross morphology, presumably through disruption of the nematode cuticle. Adult worms homozygous for rol-3(e754) exhibit rotation about their long axis associated with a left-hand twisted cuticle, musculature, gut and ventral nerve cord. Our laboratory previously isolated 12 recessive lethal alleles of rol-3. All these lethal alleles cause an arrest in development at either early or mid-larval stages, suggesting that the rol-3 gene product performs an essential developmental function. Furthermore, through the use of the heterochronic mutants lin-14 and lin-29, we have established that the expression of rol-3(e754)'s adult specific visible function is not dependent on the presence of an adult cuticle. In an attempt to understand rol-3's developmental role we sought to identify other genes whose products interact with that of rol-3. Toward this end, we generated eight EMS induced and two gamma irradiation-induced recessive suppressors of the temperature sensitive (ts) mid-larval lethal phenotype of rol-3(s1040ts). These suppressors define two complementation groups srl-1 II and srl-2 III; and, while they suppress the rol-3(s1040) lethality, they do not suppress the adult specific visible rolling phenotype. Furthermore, there is a complex genetic interaction between srl-2 and srl-1 such that srl-2(s2506) fails to complement all srl alleles tested. These results suggest that srl-1 and srl-2 may share a common function and, thus, possibly constitute members of the same gene family. Mutations in both srl-1 and srl-2 produce no obvious hermaphrodite phenotypes in the absence of rol-3(s1040ts); however, males homozygous for either srl-1 or srl-2 display aberrant tail morphology. We present evidence suggesting that the members of srl-2 are not allele specific with respect to their suppression of rol-3 lethality, and that rol-3 may act in some way to influence proper posterior morphogenesis. Finally, based on our genetic analysis of rol-3 and the srl mutations, we present a model whereby the wild-type products of the srl loci act in a concerted manner to negatively regulate the rol-3 gene.

scholarly journals A Role for NIMA in the Nuclear Localization of Cyclin B in Aspergillus nidulans

1998 ◽  
Vol 141 (7) ◽  
pp. 1575-1587 ◽  
Author(s):  
L. Wu ◽  
S.A. Osmani ◽  
P.M. Mirabito
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Nuclear Accumulation ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Synchronous Cultures ◽  
Allele Specific

NIMA promotes entry into mitosis in late G2 by some mechanism that is after activation of the Aspergillus nidulans G2 cyclin-dependent kinase, NIMXCDC2/NIMECyclin B. Here we present two independent lines of evidence which indicate that this mechanism involves control of NIMXCDC2/NIMECyclin B localization. First, we found that NIMECyclin B localized to the nucleus and the nucleus-associated organelle, the spindle pole body, in a NIMA-dependent manner. Analysis of cells from asynchronous cultures, synchronous cultures, and cultures arrested in S or G2 showed that NIMECyclin B was predominantly nuclear during interphase, with maximal nuclear accumulation in late G2. NIMXCDC2 colocalized with NIMECyclin B in G2 cells. Although inactivation of NIMA using either the nimA1 or nimA5 temperature-sensitive mutations blocked cells in G2, NIMXCDC2/NIMECyclin B localization was predominantly cytoplasmic rather than nuclear. Second, we found that nimA interacts genetically with sonA, which is a homologue of the yeast nucleocytoplasmic transporter GLE2/RAE1. Mutations in sonA were identified as allele-specific suppressors of nimA1. The sonA1 suppressor alleviated the nuclear division and NIMECyclin B localization defects of nimA1 cells without markedly increasing NIMXCDC2 or NIMA kinase activity. These results indicate that NIMA promotes the nuclear localization of the NIMXCDC2/ NIMECyclin B complex, by a process involving SONA. This mechanism may be involved in coordinating the functions of NIMXCDC2 and NIMA in the regulation of mitosis.

scholarly journals Leaf-derived ABA regulates rice seed development via a transporter-mediated and temperature-sensitive mechanism

2021 ◽  
Vol 7 (3) ◽  
pp. eabc8873
Author(s):  
Peng Qin ◽  
Guohua Zhang ◽  
Binhua Hu ◽  
Jie Wu ◽  
Weilan Chen ◽  
...  
Keyword(s):  
Seed Development ◽  
Starch Synthesis ◽  
Biological Significance ◽  
Grain Filling ◽  
Temperature Sensitive ◽  
Rice Seed ◽  
Dependent Manner ◽  
Long Distance ◽  
Long Distance Transport ◽  
Mechanistic Basis

Long-distance transport of the phytohormone abscisic acid (ABA) has been studied for ~50 years, yet its mechanistic basis and biological significance remain very poorly understood. Here, we show that leaf-derived ABA controls rice seed development in a temperature-dependent manner and is regulated by defective grain-filling 1 (DG1), a multidrug and toxic compound extrusion transporter that effluxes ABA at nodes and rachilla. Specifically, ABA is biosynthesized in both WT and dg1 leaves, but only WT caryopses accumulate leaf-derived ABA. Our demonstration that leaf-derived ABA activates starch synthesis genes explains the incompletely filled and floury seed phenotypes in dg1. Both the DG1-mediated long-distance ABA transport efficiency and grain-filling phenotypes are temperature sensitive. Moreover, we extended these mechanistic insights to other cereals by observing similar grain-filling defects in a maize DG1 ortholog mutant. Our study demonstrates that rice uses a leaf-to-caryopsis ABA transport–based mechanism to ensure normal seed development in response to variable temperatures.

Phase I Trial of the Proteasome Inhibitor PS-341 in Patients With Refractory Hematologic Malignancies

2002 ◽  
Vol 20 (22) ◽  
pp. 4420-4427 ◽  
Author(s):  
Robert Z. Orlowski ◽  
Thomas E. Stinchcombe ◽  
Beverly S. Mitchell ◽  
Thomas C. Shea ◽  
Albert S. Baldwin ◽  
...  
Keyword(s):  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Complete Response ◽  
Hematologic Malignancies ◽  
Maximum Tolerated Dose ◽  
20S Proteasome ◽  
Dependent Manner ◽  
Mantle Cell ◽  
Heavily Pretreated ◽  
Systemic Hypersensitivity

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacodynamics (PD) of the proteasome inhibitor bortezomib (previously known as PS-341) in patients with refractory hematologic malignancies.PATIENTS AND METHODS: Patients received PS-341 twice weekly for 4 weeks at either 0.40, 1.04, 1.20, or 1.38 mg/m2, followed by a 2-week rest. The PD of PS-341 was evaluated by measurement of whole blood 20S proteasome activity.RESULTS: Twenty-seven patients received 293 doses of PS-341, including 24 complete cycles. DLTs at doses above the 1.04-mg/m2MTD attributed to PS-341 included thrombocytopenia, hyponatremia, hypokalemia, fatigue, and malaise. In three of 10 patients receiving additional therapy, serious reversible adverse events appeared during cycle 2, including one episode of postural hypotension, one systemic hypersensitivity reaction, and grade 4 transaminitis in a patient with hepatitis C and a substantial acetaminophen ingestion. PD studies revealed PS-341 induced 20S proteasome inhibition in a time-dependent manner, and this inhibition was also related to both the dose in milligrams per meter squared, and the absolute dose of PS-341. Among nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing one cycle of therapy, there was one complete response and a reduction in paraprotein levels and/or marrow plasmacytosis in eight others. In addition, one patient with mantle cell lymphoma and another with follicular lymphoma had shrinkage of nodal disease.CONCLUSION: PS-341 was well tolerated at 1.04 mg/m2on this dose-intensive schedule, although patients need to be monitored for electrolyte abnormalities and late toxicities. Additional studies are indicated to determine whether incorporation of dose/body surface area yields a superior PD model to dosing without normalization. PS-341 showed activity against refractory multiple myeloma and possibly non-Hodgkin’s lymphoma in this study, and merits further investigation in these populations.

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.

Genetic exploration of interactive domains in RNA polymerase II subunits

1990 ◽  
Vol 10 (5) ◽  
pp. 1908-1914
Author(s):  
C Martin ◽  
S Okamura ◽  
R Young
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Temperature Sensitive ◽  
Suppressor Mutations ◽  
Region I ◽  
Temperature Sensitive Mutation ◽  
Allele Specific ◽  
Separate Protein

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

scholarly journals Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

1989 ◽  
Vol 9 (3) ◽  
pp. 875-884 ◽  
Author(s):  
T S Hays ◽  
R Deuring ◽  
B Robertson ◽  
M Prout ◽  
M T Fuller
Keyword(s):  
Drosophila Melanogaster ◽  
Missense Mutation ◽  
Null Allele ◽  
Genetic Interaction ◽  
Structural Proteins ◽  
Interacting Proteins ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Gene Encoding ◽  
Alpha Tubulin

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.

Sign in / Sign up

Export Citation Format

Share Document