Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The point mutation at nucleotide 323 within the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum, which distinguishes pyrimethamine-sensitive from drug-resistant isolates, can be discriminated by the polymerase chain reaction using mutation-specific primers. The technique makes use of the principle that short oligonucleotides with a perfect match at their 3' ends, complementary to the mutation to be detected, will initiate the polymerization by Taq polymerase far more efficiently than primers with a single mismatch in this position. The Asn-108 codon was detected using a primer of 17 nucleotides with an adenosine at its 3' end, the Thr-108 codon with a 14-mer primer ending with a cytosine and the Ser-108 codon with a 16-mer containing guanidine at the critical 3' end. By selecting appropriate counterprimers, the size of the amplification products is either indicative of pyrimethamine-resistant parasites of the 7G8 type, or of pyrimethamine-sensitive parasites of the FCR-3 type or 3D7 type. The fragments obtained can be easily separated in a single lane after agarose gel electrophoresis. Coded P. falciparum DNA samples were typed unambiguously using these primers as were reconstituted parasitized blood samples stored as high salt lysates. Sensitivity, speed and specificity make this assay a realistic alternative to in vitro drug testing to monitor the resistance of P. falciparum to inhibitors of the dihydrofolate reductase.
Mol Biochem Parasitol 1990 Mar
PMID:Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction. 218 8

The tsx-206 allele encodes an altered Tsx protein, Tsx-206, that can no longer function as the T6 receptor. We show here that this allele also confers resistance to the Tsx-specific phages H1, H3, H8, K9, K18 and Ox1 but not to colicin K. The Tsx-206 protein still mediates the efficient permeation of deoxyadenosine across the outer membrane at low substrate concentration. A host-range mutant of phage T6, T6h3.1, was isolated which can use both the Tsx-206 and the Tsx wild-type protein as its receptor. Cloning and DNA sequence analysis of the tsx-206 allele showed that the phage resistant phenotype was associated with an Asn to Tyr substitution at position 254 of the 272-residue Tsx protein.
Mol Gen Genet 1990 May
PMID:Analysis of a mutated phage T6 receptor protein of Escherichia coli K 12. 219 19

The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system [(1989) Methods Enzymol. 178, 497-515]. We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure [J. Mol. Biol. 190, 593-604]. In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region. These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments. Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures.
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PMID:Structural features of the McPC603 Fab fragment not defined in the X-ray structure. 222 4

Cytochrome P450c21 (steroid 21-hydroxylase) is a key enzyme in the synthesis of cortisol, whose deficiency is the cause of a common genetic disease, congenital adrenal hyperplasia. We have expressed P450c21 (steroid 21-hydroxylase) in E. coli and mammalian cells. In E. coli, P450c21 cDNA was cloned into a T7 expression vector to produce a large amount of P450c21 fusion protein, which enabled antiserum production. In mammalian cells, a plasmid containing full-length P450c21 cDNA (phc21) was constructed and transfected into COS-1 cells to produce active P450c21, which was detected by immunoblotting and 21-hydroxylase activity assay. This system was used to assay mutations involved in the disease. Ile172 of phc21 corresponding to the site of mutation in some cases of the disease was mutagenized to become Asn, Leu, His, or Gln. Mutant as well as normal P450c21 was produced when their cDNAs were transfected into COS-1 cells. The mutant proteins, however, had greatly reduced 21-hydroxylase activities. Therefore, missense mutation at Ile172 resulted in inactivation of the enzyme, but not in repression of enzyme synthesis. The Leu for Ile substitution at amino acid 172 did not result in partial restoration of enzymatic activity, indicating that hydrophobicity at this residue may not play a role in its function.
Mol Endocrinol 1990 Jun
PMID:Expression of human 21-hydroxylase (P450c21) in bacterial and mammalian cells: a system to characterize normal and mutant enzymes. 223 46

We have identified dihydrofolate reductase (DHFR) gene point mutations and chromosomal changes in pyrimethamine-resistant mutants selected in vitro of Plasmodium falciparum strain FCR3. A pyrimethamine-resistant derivative of the pyrimethamine-sensitive strain FCR3, FCR3-D8, that had been grown in the absence of pyrimethamine for an extended time, was grown in two concentrations of pyrimethamine, and surviving drug-resistant parasites were subcloned. One selected mutant, FCR3-D81, that grew at 1 X 10(-6) M pyrimethamine, contained a single point mutation in the DHFR domain which caused an amino acid change (Phe to Ser) at amino acid 223, whereas another mutant, FCR3-D85, that grew at 5 X 10(-6) M pyrimethamine had that same mutation and an additional point mutation that changed amino acid 54 (Asp to Asn). The selection of FCR3-D85, whose nucleotide sequence was identical to that previously reported for FCR3-D8, confirmed that the original FCR3-D8 parasite population had changed during extended growth in vitro in the absence of drug pressure. FCR3-D81 and FCR3-D85 cells contained different pairs of polymorphic chromosomes that hybridized to a DHFR-TS probe as well as to three other chromosome 4 specific DNAs, indicating that at least part of chromosome 4 had been duplicated and that these parasites were aneuploid with 15 rather than 14 chromosomes. The mutant DHFR-TS genes were diploid. We consider the roles of the polymorphic chromosome duplications and DHFR point mutation(s) as causes of pyrimethamine resistance.
Mol Biochem Parasitol 1990 Aug
PMID:Mutant dihydrofolate reductase-thymidylate synthase genes in pyrimethamine-resistant Plasmodium falciparum with polymorphic chromosome duplications. 223 1

Cleavage of mouse IgA T15 with papain yielded (a) a glycosylated Fab fragment, (b) a non-glycosylated Fc fragment and (c) a glycosylated C-terminal peptide. The cleavage sites at the hinge and at the end of the C alpha 3 domain were located by sequencing. The two glycopeptides were prepared from the Fab and C-terminal fragments by pronase digestion. The C alpha 1 glycopeptide at Asn 155 was complex type with alpha (1-3)galactose terminal groups, and closely resembled the Asn 171 glycopeptide of mouse IgM (Anderson et al. (1985) Arch. Biochem. Biophys. 243, 605-618). In contrast, the C-terminal glycopeptide at Asn 446 was entirely different from the corresponding IgM glycopeptide, being complex rather than high-mannose type.
Mol Immunol 1990 Nov
PMID:The glycopeptides of the mouse immunoglobulin A T15. 224 87

Dolichol-linked oligosaccharides consisting of two N-acetylglucosamine, nine mannose, and three glucose residues (Glc3Man9GlcNAc2) are transferred to proteins that contain the consensus sequence Asn-X-Ser/Thr. This transfer occurs upon protein import into the lumen of the endoplasmic reticulum. An intermediate in the biosynthesis of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide contains two GlcNAc and five mannose residues. This intermediate serves as a substrate for further mannosylation and glucosylation before transfer to protein. The addition of the sixth mannose residue to this intermediate requires the enzyme mannosyltransferase VI and the mannose donor, mannose-P-dolichol. Several different CHO cell line mutants that fail to efficiently catalyze this transfer have been described. In this report, we examine seven independent mutant cell lines with various biochemical phenotypes and demonstrate that all can be assigned to one of three genetic complementation groups. One mutation affects mannose-P-dolichol biosynthesis (Lec15), three affect dolichol phosphate biosynthesis (Lec9), and three appear to affect the functional orientation of enzyme substrates (PIR).
Somat Cell Mol Genet 1990 Nov
PMID:Complementing mutant alleles define three loci involved in mannosylation of Man5-GlcNAc2-P-P-dolichol in Chinese hamster ovary cells. 226 28

Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to beta-tubulin. The benA gene of three independently isolated rhizoxin-resistant (Rhir) mutants of Aspergillus nidulans was cloned, sequenced and compared with that of the wild-type, rhizoxin-sensitive (Rhis) strain. In all three Rhir mutants, the AAC codon for Asn-100 of the benA beta-tubulin gene was altered to ATC, coding for Ile. Sequence displacement experiments confirmed that the substitution of Ile for Asn-100 confers resistance to rhizoxin in this organism. The amino acid sequences of beta-tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions. The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are naturally occurring Rhir organisms whose beta-tubulin genes encode Ile and Val respectively at the 100th amino acid residue. The Ile-100 of S. pombe and the Val-100 of S. cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques. The resultant haploid strains of these two yeasts uniquely expressing beta-tubulin (Asn-100) instead of beta-tubulin (Ile-100 or Val-100) were found to be Rhis. Haploid yeast expressing beta-tubulin (Asn-100) is normal except for its sensitivity to rhizoxin. These results suggest that rhizoxin resistance has a common basis in both naturally occurring species and experimentally selected mutants in the substitution of Ile or Val for Asn-100 in beta-tubulin.
Mol Gen Genet 1990 Jul
PMID:Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin. 227 23

Possible effects of single point mutations on the ligand-binding capabilities of human serum albumin (Alb) were investigated by studying the interactions between the strongly bound drugs warfarin, salicylate, and diazepam and five structurally characterized genetic variants of the protein. Equilibrium dialysis data, obtained with the variants and normal serum Alb, revealed pronounced reductions in high affinity binding of all three ligands to Alb Canterbury (313 Lys----Asn) and to Alb Parklands (365 Asp----His). By contrast, unchanged binding of the drugs was found in the case of Alb Verona (570 Glu----Lys). Different effects on binding were observed for the other two variants. Salicylate was the only drug bound with a lower affinity to Alb Niigata (269 Asp----Gly), whereas binding of both salicylate and diazepam to Alb Roma (321 Glu----Lys) were moderately reduced. In about half of the cases of diminished binding, the primary association constant was reduced by 1 order of magnitude, giving rise to an increase in the unbound fraction of the drugs of 500% or more at therapeutically relevant molar ratios of drug and protein. Changes in protein charge seem to be of only minor importance for reduced binding. More likely, conformational changes in the 313-365 region of the proteins are the main cause for diminished binding of these diverse ligands, which probably have different high affinity binding sites. The specific reduction in salicylate binding after modification of residue 269 may be due to conformational changes at or close to the salicylate binding site.
Mol Pharmacol 1990 Feb
PMID:Binding of warfarin, salicylate, and diazepam to genetic variants of human serum albumin with known mutations. 230 52

The 16,775 base-pair mitochondrial genome of the white Leghorn chicken has been cloned and sequenced. The avian genome encodes the same set of genes (13 proteins, 2 rRNAs and 22 tRNAs) as do other vertebrate mitochondrial DNAs and is organized in a very similar economical fashion. There are very few intergenic nucleotides and several instances of overlaps between protein or tRNA genes. The protein genes are highly similar to their mammalian and amphibian counterparts and are translated according to the same variant genetic code. Despite these highly conserved features, the chicken mitochondrial genome displays two distinctive characteristics. First, it exhibits a novel gene order, the contiguous tRNA(Glu) and ND6 genes are located immediately adjacent to the displacement loop region of the molecule, just ahead of the contiguous tRNA(Pro), tRNA(Thr) and cytochrome b genes, which border the displacement loop region in other vertebrate mitochondrial genomes. This unusual gene order is conserved among the galliform birds. Second, a light-strand replication origin, equivalent to the conserved sequence found between the tRNA(Cys) and tRNA(Asn) genes in all vertebrate mitochondrial genomes sequenced thus far, is absent in the chicken genome. These observations indicate that galliform mitochondrial genomes departed from their mammalian and amphibian counterparts during the course of evolution of vertebrate species. These unexpected characteristics represent useful markers for investigating phylogenetic relationships at a higher taxonomic level.
J Mol Biol 1990 Apr 20
PMID:Sequence and gene organization of the chicken mitochondrial genome. A novel gene order in higher vertebrates. 232 78


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