scholarly journals Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase fused to diphtheria toxin

1996 ◽  
Vol 313 (2) ◽  
pp. 647-653 ◽  
Author(s):  
Olav KLINGENBERG ◽  
Sjur OLSNES
Keyword(s):  
Fusion Protein ◽  
Dihydrofolate Reductase ◽  
Diphtheria Toxin ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Toxin B ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Diphtheria Toxin A

A fusion protein consisting of dihydrofolate reductase and diphtheria toxin A-fragment was made by genetically linking cDNA for the two proteins followed by in vitro transcription and translation in a rabbit reticulocyte lysate system. The dihydrofolate reductase in the fusion protein exhibited enzyme activity and, in the presence of methotrexate which imposes a tight structure on dihydrofolate reductase, it was trypsin resistant, indicating that it was correctly folded. When reconstituted with diphtheria toxin B-fragment, it bound specifically to diphtheria toxin receptors and was translocated into cells upon exposure to low pH. Methotrexate prevented the translocation. Protein synthesis was inhibited in cells incubated with the reconstituted fusion protein, but the inhibition was reduced in the presence of methotrexate. We also made a fusion protein containing a mutated dihydrofolate reductase with much lower affinity to methotrexate. Methotrexate did not prevent translocation of this protein. The data indicate that methotrexate prevents translocation of the fusion protein containing wild-type dihydrofolate reductase by imposing a tight structure on to the enzyme.

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).

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 GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis

1993 ◽  
Vol 13 (12) ◽  
pp. 7232-7238
Author(s):  
W D Rapp ◽  
D S Lupold ◽  
S Mack ◽  
D B Stern
Keyword(s):  
Promoter Region ◽  
Consensus Sequence ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Transcription Start ◽  
Transcription System ◽  
Mitochondrial Promoters ◽  
Point Mutagenesis

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

scholarly journals Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

1987 ◽  
Vol 7 (4) ◽  
pp. 1576-1579 ◽  
Author(s):  
F Maxwell ◽  
I H Maxwell ◽  
L M Glode
Keyword(s):  
Diphtheria Toxin ◽  
Predicted Amino Acid Sequence ◽  
Specific Cell ◽  
Wild Type ◽  
Transient Expression Assay ◽  
Toxin A ◽  
Coding Sequence ◽  
Chain Coding ◽  
Diphtheria Toxin A ◽  
A Chain

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.

scholarly journals Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

1993 ◽  
Vol 293 (1) ◽  
pp. 181-185 ◽  
Author(s):  
N J Watkins ◽  
A K Campbell
Keyword(s):  
Light Emission ◽  
Specific Activity ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Proline Residue ◽  
Long Term Stability ◽  
Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

scholarly journals Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051 ◽  
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

scholarly journals Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid

2010 ◽  
Vol 54 (9) ◽  
pp. 3776-3782 ◽  
Author(s):  
Feng Wang ◽  
Paras Jain ◽  
Gulcin Gulten ◽  
Zhen Liu ◽  
Yicheng Feng ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dihydrofolate Reductase ◽  
Fold Increase ◽  
Antitubercular Activity ◽  
Experimental Sample ◽  
Wild Type ◽  
Negative Results ◽  
Sequencing Studies ◽  
Type Strains

ABSTRACT Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.

scholarly journals Conserved C-Terminal Threonine of Hepatitis C Virus NS3 Regulates Autoproteolysis and Prevents Product Inhibition

2004 ◽  
Vol 78 (2) ◽  
pp. 700-709 ◽  
Author(s):  
Wenyan Wang ◽  
Frederick C. Lahser ◽  
MinKyung Yi ◽  
Jacquelyn Wright-Minogue ◽  
Ellen Xia ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
In Vitro Transcription ◽  
Product Inhibition ◽  
Cleavage Sites ◽  
Wild Type ◽  
Genome Sequences ◽  
Mutant Proteins ◽  
C Terminus

ABSTRACT Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.

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