SEARCH · PubMed Health

Results for “bud mutant”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed2006

Proteomic identification of potential target proteins regulated by an ASK1-mediated proteolysis pathway.

The ASK1 (ARABIDOPSIS SKP1-LIKE) protein is a critical component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate proteins that are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers, we compared the proteomes of the Arabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis (2-DE). Ten protein spots with higher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to further mass spectrometry (MS) analysis. The results showed that they were proteins involved in photomorphogenesis, circadian oscillation, post-translation process, stress-responses and cell expansion or elongation, suggesting that those processes were affected in the ask1 mutant. The transcript levels of these genes were also compared based on the Affymetrix gene chip microarray data. No significant difference was observed for most of the genes, suggesting that the proteins with elevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASK1-mediated proteolysis pathway. These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes and also demonstrate the importance and necessity of studying protein levels with respect to gene functions.

Arabidopsis↗

PubMed2003

The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast.

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.

Actins↗

PubMed1993

Mutational analysis of Moloney murine leukaemia virus surface protein gp70.

Ten mutations were generated in the env gene of Moloney murine leukaemia virus DNA. The mutations were made by site-directed mutagenesis to alter basic amino acids (lysine or arginine) in the surface glycoprotein gp70. Mutants were investigated following transfection into NIH/3T3 cells. All 10 mutants released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression or virion budding. Two mutants were positive in XC plaque assay, reverse transcriptase assay and re-infection experiments, showing that these mutations occurred in parts of the molecule not essential for infection. Three mutants were negative in both the XC plaque assay and re-infection experiments, suggesting that they make non-infectious virus particles. The results indicate a defect in the early phase of infection, perhaps in receptor binding or in the fusion of virion and host membranes. The other mutations resulted in reduced infectivity of released virion particles.

3T3 Cells↗

PubMed2005

Identification of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae.

Hypoxia activates all components of the unfolded protein response (UPR), a stress response initiated by the accumulation of unfolded proteins within the endoplasmic reticulum (ER). Our group and others have shown previously that the UPR, a hypoxia-inducible factor-independent signaling pathway, mediates cell survival during hypoxia and is required for tumor growth. Identifying new genes and pathways that are important for survival during ER stress may lead to the discovery of new targets in cancer therapy. Using the set of 4,728 homozygous diploid deletion mutants in budding yeast, Saccharomyces cerevisiae, we did a functional screen for genes that conferred resistance to ER stress-inducing agents. Deletion mutants in 56 genes showed increased sensitivity under ER stress conditions. Besides the classic UPR pathway and genes related to calcium homeostasis, we report that two additional pathways, including the SLT2 mitogen-activated protein kinase (MAPK) pathway and the osmosensing MAPK pathway, were also required for survival during ER stress. We further show that the SLT2 MAPK pathway was activated during ER stress, was responsible for increased resistance to ER stress, and functioned independently of the classic IRE1/HAC1 pathway. We propose that the SLT2 MAPK pathway is an important cell survival signaling pathway during ER stress. This study shows the feasibility of using the yeast deletion pool to identify relevant mammalian orthologues of the UPR.

Basic-Leucine Zipper Transcription Factors↗

PubMed1997

Shh, Fgf4 and Hoxd gene expression in the mouse limb mutant hypodactyly.

The semidominant mouse mutation hypodactyly (Hd), caused by a deletion within the Hoxa13 gene, results in reduced digits; heterozygotes lack digit I in the hindlimb and homozygotes have only one digit on each limb. We investigated expression of Shh and Fgf4 signaling molecules involved in digit specification in mutant limb buds. Shh and Fgf4 are expressed in the posterior part of the limb buds as normal but expression may be slightly prolonged. The extent of digit reduction in hypodactyly is much more severe than in the Hoxa13 deficient mouse and resembles that in the Hoxa13(-/-)/Hoxd13(-/-) double mutant mouse. We found that the pattern of Hoxd13 and Hoxd11 transcripts was not markedly different in the mutant compared with the normal limbs even though the mutant limbs are narrower. Therefore Hoxd genes are transcribed as normal in the mutant. This makes it likely that the severe digit reductions in hypodactyly are caused by interference with Hoxd13 function at the protein level. Similar interactions between mutant and normal HOX gene products have been suggested to occur in the human semidominant disorder, synpolydactyly, caused by mutations in HOXD13.

Animals↗

PubMed1996

Analysis of limb patterning in BMP-7-deficient mice.

Bone morphogenetic proteins (BMPs) are polypeptide signaling molecules, belonging to the TGF-beta superfamily. They were originally identified by their ability to induce ectopic bone formation, but their expression patterns in embryos suggest multiple functions. BMP-7-deficient mice show among other mesodermal and skeletal patterning defects, polydactyly in the hindlimbs [Luo G, Hofmann C, Bronckers ALJJ, Sohocki M, Bradley A, Karsenty G (1995): Genes Dev 9:2808-2820; Dudley AT, Lyons KM, Robertson EJ (1995): Genes Dev 9:2795-2807]. Here we report a more detailed analysis of the limb phenotype in BMP-7-deficient mice using in situ hybridization to monitor expression of molecules implicated in patterning processes of the developing vertebrate limb. In previous studies we showed that Sonic hedgehog (Shh) was expressed normally, but Hoxd-13 expression in limb mesenchyme was lower in BMP-7 mutant limbs. Here we show that Hoxd-11 expression domains are also contracted and decreased in intensity in mutant limbs, suggesting that 5' genes of the Hoxd cluster are coordinately downregulated, while another Bmp, Bmp-2, which can be activated by Shh, is similarly expressed. The mutant limb buds are broader than normal buds, and fibroblast growth factor Fgf-8 is expressed throughout the extended ridge. However, expression of the homeobox gene Msx-1, which has been shown to be involved in epithelial-mesenchymal interactions during limb development, was decreased in the mesenchyme of BMP-7 mutant limbs. Taken together, our data suggest that BMP-7 is involved in regulating proliferation and/or epithelial-mesenchymal interactions in the developing limb.

Animals↗

PubMed2000

Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae.

We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This receptor, along with SRP, targets ribosome-nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.

Actins↗

PubMed2006

A luciferase-based budding assay for Ebola virus.

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.

Cell Line↗

PubMed1988

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant.

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.

Animals↗

PubMed2000

dackel acts in the ectoderm of the zebrafish pectoral fin bud to maintain AER signaling.

Classical embryological studies have implied the existence of an apical ectodermal maintenance factor (AEMF) that sustains signaling from the apical ectodermal ridge (AER) during vertebrate limb development. Recent evidence suggests that AEMF activity is composed of different signals involving both a sonic hedgehog (Shh) signal and a fibroblast growth factor 10 (Fgf10) signal from the mesenchyme. In this study we show that the product of the dackel (dak) gene is one of the components that acts in the epidermis of the zebrafish pectoral fin bud to maintain signaling from the apical fold, which is homologous to the AER of tetrapods. dak acts synergistically with Shh to induce fgf4 and fgf8 expression but independently of Shh in promoting apical fold morphogenesis. The failure of dak mutant fin buds to progress from the initial fin induction phase to the autonomous outgrowth phase causes loss of both AER and Shh activity, and subsequently results in a proximodistal truncation of the fin, similar to the result obtained by ridge ablation experiments in the chicken. Further analysis of the dak mutant phenotype indicates that the activity of the transcription factor engrailed 1 (En1) in the ventral non-ridge ectoderm also depends on a maintenance signal probably provided by the ridge. This result uncovers a new interaction between the AER and the dorsoventral organizer in the zebrafish pectoral fin bud.

Animals↗

PubMed1986

Vascular system in the developing wing bud of normal and talpid mutant chick embryos.

The vascular limb mesoderm probably plays a prominent role in limb pattern formation in both normal and talpid chick embryos. The differential vascularization, or its consequences, is a factor controlling the initial stages of differentiation in the developing bud. The axial artery runs from the subclavian artery to the distal region of the normal limb bud, whereas in the talpid3 only secondary blood vessels develop. In the talpid, the gene permits the chondrogenic regions to grow and at the same time keeps the peripheral regions to a certain size. The mesenchyme tissue lies within the effective range of a metabolic gradient extending from either the ectodermal surface or the peripheral vessels to the limb axis.

Animals↗

PubMed1989

The limb deformity gene is required for apical ectodermal ridge differentiation and anteroposterior limb pattern formation.

To gain insight into the role of the limb deformity (ld) gene in limb morphogenesis, we examined the morphologic details of early embryonic limb formation in the mutant ld/ld mouse. Initial morphological differences between wild-type and homozygous ld embryos are apparent during early gestational day 10, a time period during which anteroposterior limb morphogenesis occurs. As a result of a shortened anteroposterior axis, the mutant limb bud appears more pointed than its wild-type counterpart. In addition, the apical ectodermal ridge (AER), a structure crucial to both proximodistal and anteroposterior limb development, fails to differentiate properly in mutant ld embryos. Consistent with these observations, molecular analysis of the limb promordia shows that the limb ectoderm contains a level of ld transcripts fivefold higher relative to its mesenchyme. Furthermore, expression of ld transcripts in other parts of the developing embryo and in primitive streak embryos (gestational day 7) suggests possible roles for this gene in the earliest determinative events of morphogenesis. These data lead us to conclude that ld gene products are required for both proper AER differentiation and anteroposterior pattern formation in limb mesenchyme.

Animals↗

PubMed1994

A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments.

Rab1 is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rab1 we have analyzed the function of a mutant (rab1a[S25N]) containing a substitution which perturbs Mg2+ coordination and reduces the affinity for GTP, resulting in a form which is likely to be restricted to the GDP-bound state. The rab1a(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rab1 during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rab1 function. Both rab1a and rab1b (92% identity) were able to antagonize the inhibitory activity of the rab1a(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rab1 mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rab1 function in vesicular traffic.

Base Sequence↗

PubMed2004

Function of BMPs in the apical ectoderm of the developing mouse limb.

Several bone morphogenetic proteins (BMPs) are expressed in the apical ectodermal ridge (AER), a critical signaling center that directs the outgrowth and patterning of limb mesoderm, but little is known about their function. To study the functions of apical ectodermal BMPs, an AER-specific promoter element from the Msx2 gene was used to target expression of the potent BMP antagonist noggin to the apical ectoderm of the limbs of transgenic mice. Msx2-noggin mutant mice have severely malformed limbs characterized by syndactyly, postaxial polydactyly, and dorsal transformations of ventral structures indicated by absence of ventral footpads and presence of supernumerary ventral nails. Mutant limb buds exhibit a dorsoventral (DV) and anteroposterior (AP) expansion in the extent of the AER. AER activity persists longer than normal and is maintained in regions of the apical ectoderm where its activity normally ceases. Mutant limbs possess a broad band of mesodermal tissue along the distal periphery that is absent from normal limbs and which fails to undergo the apoptosis that normally occurs in the subectodermal mesoderm. Taken together, our results suggest that apical ectodermal BMPs may delimit the boundaries of the AER by preventing adjacent nonridge ectodermal cells from becoming AER cells; negatively modulate AER activity and thus fine-tune the strength of AER signaling; and regulate the apoptosis of the distal subectodermal mesoderm that occurs as AER activity attenuates, an event that is essential for normal limb development. Our results also confirm that ectodermal BMP signaling regulates DV patterning.

Animals↗

PubMed2004

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27.

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

Amino Acid Sequence↗

PubMed1994

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously ts and cold-sensitive (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.

Base Sequence↗

PubMed2003

A role for the mesenchymal T-box gene Brachyury in AER formation during limb development.

During limb development, several signaling centers organize limb pattern. One of these, the apical ectodermal ridge (AER), is critical for proximodistal limb outgrowth mediated by FGFs. Signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. Ectodermal factors, particularly En1, play a critical role in regulating morphogenesis of a mature, compact AER along the distal limb apex, from a broad ventral ectodermal precursor domain. Contribution of mesodermal factors to the morphogenesis of a mature AER is less clear. We previously noted that the chick T gene (Brachyury), the prototypical T-box transcription factor, is expressed in the limb bud as well as axial mesoderm and primitive streak. Here we show that T is expressed in lateral plate mesoderm at the onset of limb bud formation and subsequently in the subridge mesoderm beneath the AER. Retroviral misexpression of T in chick results in anterior extension of the AER and subsequent limb phenotypes consistent with augmented AER extent and function. Analysis of markers for functional AER in mouse T(-/-) null mutant limb buds reveals disrupted AER morphogenesis. Our data also suggest that FGF and WNT signals may operate both upstream and downstream of T. Taken together, the results show that T plays a role in the regulation of AER formation, particularly maturation, and suggest that T may also be a component of the epithelialmesenchymal regulatory loop involved in maintenance of a mature functioning AER.

Animals↗

PubMed1990

Altered budding site of a pantropic mutant of Sendai virus, F1-R, in polarized epithelial cells.

A protease activation mutant of Sendai virus, F1-R, causes a systemic infection in mice, whereas wild-type virus is exclusively pneumotropic (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). Budding of F1-R has been observed bidirectionally at the apical and basolateral surfaces of the bronchial epithelium of mice and of MDCK cells, whereas wild-type virus buds apically (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J. T. Seto, J. Virol. 64:3627-3634, 1990). In this study, wild-type virus was shown to be produced primarily from the apical site of polarized MDCK cells grown on permeable membrane filters. Surface immunofluorescence and immunoprecipitation analyses revealed that transmembrane glycoproteins HN and F were expressed predominantly at the apical domain of the plasma membrane. On the other hand, infectious progeny of F1-R was released from the apical and basolateral surfaces, and HN and F were expressed at both regions of the cells. Since F1-R has amino acid substitutions in F and M proteins but none in HN, the altered budding of the virus and transport of the envelope glycoproteins might be attributed to interactions by F and M proteins. These findings suggest that in addition to proteolytic activation of the F glycoprotein, the differential site of budding, at the primary target of infection, is a determinant for organ tropism of Sendai virus in mice.

Animals↗