scholarly journals Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

1990 ◽  
Vol 10 (6) ◽  
pp. 2801-2808 ◽  
Author(s):  
D T Mooney ◽  
D B Pilgrim ◽  
E T Young
Keyword(s):  
Point Mutations ◽  
Wild Type ◽  
Urea Treatment ◽  
Amino Terminal ◽  
In Vitro Processing ◽  
The Matrix ◽  
Terminal Amino ◽  
Processing Protease

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease

1990 ◽  
Vol 10 (6) ◽  
pp. 2801-2808
Author(s):  
D T Mooney ◽  
D B Pilgrim ◽  
E T Young
Keyword(s):  
Point Mutations ◽  
Wild Type ◽  
Urea Treatment ◽  
Amino Terminal ◽  
In Vitro Processing ◽  
The Matrix ◽  
Terminal Amino ◽  
Processing Protease

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct 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 Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Overexpression of the Matrix Metalloproteinase Matrilysin Results in Premature Mammary Gland Differentiation and Male Infertility

1998 ◽  
Vol 9 (2) ◽  
pp. 421-435 ◽  
Author(s):  
Laura A. Rudolph-Owen ◽  
Paul Cannon ◽  
Lynn M. Matrisian
Keyword(s):  
Matrix Metalloproteinase ◽  
Transgenic Animals ◽  
Reproductive Organs ◽  
Mammary Development ◽  
Seminiferous Tubules ◽  
Interstitial Tissue ◽  
Wild Type ◽  
The Matrix

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce β-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Specific protein binding to the simian virus 40 enhancer in vitro.

1986 ◽  
Vol 6 (6) ◽  
pp. 2098-2105 ◽  
Author(s):  
A G Wildeman ◽  
M Zenke ◽  
C Schatz ◽  
M Wintzerith ◽  
T Grundström ◽  
...  
Keyword(s):  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Specific Protein ◽  
Dnase I ◽  
Wild Type ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

2016 ◽  
Vol 39 (2) ◽  
pp. 544-553 ◽  
Author(s):  
Sabrina V. Martini ◽  
Adriana L. Silva ◽  
Debora Ferreira ◽  
Rafael Rabelo ◽  
Felipe M. Ornellas ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Point Mutations ◽  
Lung Mechanics ◽  
Mouse Lung ◽  
Control Group ◽  
Polymorphonuclear Cells ◽  
Wild Type ◽  
Tyrosine Residues

Background/Aims: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

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