scholarly journals Dominant Gain-of-Function Mutations in Hsp104p Reveal Crucial Roles for the Middle Region

2004 ◽  
Vol 15 (5) ◽  
pp. 2061-2072 ◽  
Author(s):  
Eric C. Schirmer ◽  
Oliver R. Homann ◽  
Anthony S. Kowal ◽  
Susan Lindquist
Keyword(s):  
Cell Growth ◽  
Point Mutations ◽  
Middle Region ◽  
Wild Type ◽  
Tight Control ◽  
Extreme Stress ◽  
Amino Acid Stretch ◽  
Protein Remodeling ◽  
Isolated Point ◽  
Bud Morphology

Heat-shock protein 104 (Hsp104p) is a protein-remodeling factor that promotes survival after extreme stress by disassembling aggregated proteins and can either promote or prevent the propagation of prions (protein-based genetic elements). Hsp104p can be greatly overexpressed without slowing growth, suggesting tight control of its powerful protein-remodeling activities. We isolated point mutations in Hsp104p that interfere with this control and block cell growth. Each mutant contained alterations in the middle region (MR). Each of the three MR point mutations analyzed in detail had distinct phenotypes. In combination with nucleotide binding site mutations, Hsp104pT499Ialtered bud morphology and caused septin mislocalization, colocalizing with the misplaced septins. Point mutations in the septin Cdc12p suppressed this phenotype, suggesting that it is due to direct Hsp104p–septin interactions. Hsp104pA503Vdid not perturb morphology but stopped cell growth. Remarkably, when expressed transiently, the mutant protein promoted survival after extreme stress as effectively as did wild-type Hsp104p. Hsp104pA509Dhad no deleterious effects on growth or morphology but had a greatly reduced ability to promote thermotolerance. That mutations in an 11-amino acid stretch of the MR have such profound and diverse effects suggests the MR plays a central role in regulating Hsp104p function.

Targeting BCR-ABL and Its Downstream Signaling Cascade as Therapy for Chronic Myelogenous Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2964-2964 ◽  
Author(s):  
Nicholas J. Donato ◽  
Ji Wu ◽  
Ling Y. Kong ◽  
Feng Meng ◽  
Waldemar Priebe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Tyrosine Kinase ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Therapeutic Activity ◽  
Wild Type ◽  
Growth And Survival ◽  
Downstream Signaling ◽  
Kinase Inhibitory Activity

Abstract BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of chromosomal translocation in chronic myelogenous leukemia (CML). The tyrosine kinase activity of BCR-ABL activates signaling cascades that induce cytokine independence and transformation of myeloid progenitor cells. Targeted inhibition of this kinase with specific inhibitors (imatinib or BMS-354825) is a very effective therapy for some CML patients but resistance to these agents (through point mutations and other mechanisms) leads to advanced disease with very few therapeutic options. An alternate therapeutic strategy is to reduce BCR-ABL expression or its critical downstream signaling elements important for transformation. We examined BCR-ABL signaling elements and gene expression changes that occur in CML cells following kinase inhibition by imatinib in newly established imatinib sensitive and resistant cells to identify critical signaling elements involved in CML cell death. Imatinib rapidly and progressively suppressed c-myc expression in imatinib sensitive but not resistant cells prior to the onset of apoptosis. These results suggested that c-myc expression was regulated by BCR-ABL signaling and may play a role in CML tumorigenicity. To confirm a role for c-myc in CML cell growth and/or survival, c-myc expression was specifically down-regulated by siRNA using a novel electroporation instrument (AMAXA) that permits high level gene transfer with limited toxicity in CML cell lines. Jak2 siRNA was used as a control. c-myc, but not Jak2 siRNA, suppressed c-myc expression and cell growth and survival in both imatinib sensitive and resistant CML cells, suggesting that targeted suppression of c-myc may have therapeutic activity against both kinase inhibitor sensitive and resistant CML cells. Since the tyrphostin AG490 was previously shown to inhibit c-myc expression in CML cells through its inhibitory effects on Jak2, we screened a series of > 200 AG490 derivatives for their ability to rapidly reduce c-myc expression in hematological malignancies. After several rounds of testing we synthesized an agent (WP-1066) capable of rapid c-myc downregulation (beginning 1–5 min after treatment with 1–2 microM) but poor Jak2 kinase inhibitory activity (IC50 > 100 microM). These results suggested a more direct effect of WP-1066 on c-myc protein expression than AG490 and mechanistic studies suggest that WP-1066 reduces c-myc protein stability but does not affect c-myc gene expression. In BCR-ABL expressing cells WP-1066 rapidly reduced c-myc protein levels in CML cells and inhibited the growth and survival of cell lines or patient specimens expressing wild-type or mutant forms of BCR-ABL that effect tyrosine kinase inhibitory activity (T315I in BV-173R cells). Equal concentrations of imatinib or WP-1066 reduced BCR-ABL activation and downstream signaling (Stat5 phosphorylation) in CML cells. However, WP-1066 differed from imatinib in its ability to downregulate BCR-ABL protein expression without affects on c-abl or Stat5 expression. Similar results were obtained in clinical specimens taken from patients with BCR-ABL point mutations that mediate imatinib (or BMS-354825) resistance. Nude mouse studies demonstrated that WP-1066 reduced the growth of K562 tumors to an extent similar to that of imatinib. Together these results suggest that WP-1066 downregulates BCR-ABL and c-myc expression, induces apoptosis in CML cells expressing wild-type or mutant BCR-ABL and may have therapeutic activity in imatinib (or BMS-354825) resistant CML tumors.

scholarly journals CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽  
1975 ◽  
Vol 80 (4) ◽  
pp. 667-678
Author(s):  
Mary Lee S Ledbetter ◽  
Rollin D Hotchkiss
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Chromosomal Marker ◽  
Structural Abnormality ◽  
C Cells ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Low Efficiency ◽  
Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

scholarly journals A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants

2021 ◽  
Author(s):  
Myat T. Lin ◽  
Douglas J. Orr ◽  
Dawn Worrall ◽  
Martin A. J. Parry ◽  
Elizabete Carmo‐Silva ◽  
...  
Keyword(s):  
Large Subunit ◽  
Point Mutations ◽  
Subunit Gene ◽  
Wild Type

scholarly journals Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

1998 ◽  
Vol 42 (1) ◽  
pp. 164-169 ◽  
Author(s):  
A. Nzila-Mounda ◽  
E. K. Mberu ◽  
C. H. Sibley ◽  
C. V. Plowe ◽  
P. A. Winstanley ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Dihydrofolate Reductase ◽  
Drug Response ◽  
Point Mutations ◽  
Distinct Group ◽  
Wild Type ◽  
Field Isolates ◽  
Vitro Data ◽  
In Vitro Data

ABSTRACT Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P< 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1345-1353
Author(s):  
Amber K Bowers ◽  
Jennifer A Keller ◽  
Susan K Dutcher
Keyword(s):  
Molecular Markers ◽  
Chlamydomonas Reinhardtii ◽  
Candidate Genes ◽  
Splice Site ◽  
Genomic Dna ◽  
Ribosomal Proteins ◽  
Genomic Sequence ◽  
Point Mutations ◽  
Acceptor Site ◽  
Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

Abstract 3640: Mitochondrial Uncoupling Protein 2 Facilitates Endothelial Cell Growth and Angiogenesis via Reduction of Mitochondrial Superoxide

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yukio Shimasaki ◽  
Kai Chen ◽  
John F Keaney
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Mitochondrial Function ◽  
Endothelial Cell Proliferation ◽  
Wild Type ◽  
Sprouting Angiogenesis ◽  
Mitochondrial Superoxide ◽  
Endothelial Cell Growth ◽  
Aortic Explants

Background: Growing evidence suggests that mitochondrial function contributes to cell phenotype. One important component of mitochondrial function is the membrane potential that is controlled, in part, by uncoupling proteins (UCPs). Based on our previous data, the UCP2 is predominantly expressed in cultured endothelial cells. Therefore, we sought to examine the role of UCP2 in endothelial cell growth and angiogenesis. Methods and Results: Murine lung endothelial cells (MLECs) were isolated from UCP2-null and wild-type mice. UCP2-null cells were found less proliferative than wild-type cells (P<0.02, UCP2-null cells vs. wild-type cells, n=4). This defect of UCP2-null cells was rescued by UCP2 adenovirus transfection (19% increase, p<0.02 vs. LacZ adenovirus treated cells, n=3), and also rescued by transfection with manganese superoxide dismutase (MnSOD) adenovirus (53% increase, P<0.002 vs. LacZ adenovirus treated cells, n=3). We found a reciprocal relation such as no UCP2 expression and higher mitochondrial superoxide level in the MLECs (P<0.005, UCP2-null cells vs. wild-type cells, n=3), suggesting that mitochondrial superoxide may regulate endothelial cell growth. Then, we prepared murine aortic rings from UCP2-null and wild-type mice and embedded in rat tail collagen gel. The sprouting angiogenesis of UCP2-null explants was significantly less than wild-type explants (P<0.02, UCP2-null explants vs. wild-type explants, n=3– 4). Furthermore, MLECs from MnSOD-heterozygous mice showed less proliferation with lower expression of UCP2 protein and higher mitochondrial superoxide level compared to the MLECs from wild-type littermates (P<0.02, MnSOD-heterozygous cells vs. wild-type cells, n=4 – 8). We also observed less sprouting angiogenesis in MnSOD-heterozygous aortic explants than wild-type aortic explants (P<0.05, MnSOD-heterozygous explants vs. wild-type explants, n=3– 6). Conclusions: These data indicate that mitochondrial superoxide controls endothelial cell proliferation and angiogenesis, suggesting that mitochondrial metabolism modulates the endothelial cell growth and angiogenesis.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

1989 ◽  
Vol 9 (10) ◽  
pp. 4467-4472
Author(s):  
M Altmann ◽  
N Sonenberg ◽  
H Trachsel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Apparent Temperature ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Free System ◽  
Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

scholarly journals Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

Sign in / Sign up

Export Citation Format

Share Document