Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides

ABSTRACT A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia colirepeatedly selected for sulfathiazole resistance was mapped tofolP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C→T transition resulted in a Pro→Ser substitution at amino acid position 64. Replacement of the mutantfolP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance,sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by thesepurU strains.

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Cloning and characterization of Fc alpha Rb, a novel Fc alpha receptor (CD89) isoform expressed in eosinophils and neutrophils

Blood ◽

10.1182/blood.v88.11.4229.4229 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4229-4238 ◽

Cited By ~ 2

Author(s):

TB van Dijk ◽

M Bracke ◽

E Caldenhoven ◽

JA Raaijmakers ◽

JW Lammers ◽

...

Keyword(s):

Amino Acids ◽

Phosphatidyl Inositol ◽

Surface Expression ◽

Cross Linking ◽

Wild Type ◽

Human Eosinophil ◽

Transmembrane Glycoprotein ◽

Membrane Bound ◽

Intracellular Region

Abstract The Fc receptor for IgA (Fc alpha R, CD89) is a transmembrane glycoprotein found on monocytes, macrophages, neutrophils, and eosinophils. Here we describe the characterization of a novel isoform of the Fc alpha R cloned from a human eosinophil cDNA library. This clone, Fc alpha Rb, lacks the exon encoding the transmembrane/intracellular region of wild type Fc alpha R, which is replaced by 23 new amino acids. Expression of Fc alpha Rb mRNA could be detected in eosinophils and neutrophils. IIA1.6 murine pro-B cells transfected with Fc alpha Rb cDNA secrete high levels of the protein, but also a substantial amount of Fc alpha Rb is expressed at the cell membrane. Membrane-bound Fc alpha Rb binds IgA-coated beads equally well as wild type Fc alpha R. Surface expression is not affected by phosphatidyl inositol phospholipase C, indicating that glycosyl phosphatidyl inositol-linkage of Fc alpha Rb is not likely. In IIA1.6 cells expressing Fc alpha Rb and FcR gamma, which is necessary for signal transduction by wild type Fc alpha R, no tyrosine phosphorylation or Ca(2+)-mobilization could be observed after receptor cross-linking. These results indicate that Fc alpha Rb is likely to have a different function than wild-type Fc alpha R receptor.

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Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.815 ◽

1994 ◽

Vol 14 (1) ◽

pp. 815-821 ◽

Cited By ~ 21

Author(s):

P Poullet ◽

B Lin ◽

K Esson ◽

F Tamanoi

Keyword(s):

Amino Acids ◽

Neurofibromatosis Type ◽

Type I ◽

Ras Proteins ◽

Wild Type ◽

Conserved Residues ◽

Mutant Proteins ◽

Quantitative Analyses ◽

Intrinsic Gtpase Activity

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.

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Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.815-821.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 815-821

Author(s):

P Poullet ◽

B Lin ◽

K Esson ◽

F Tamanoi

Keyword(s):

Amino Acids ◽

Neurofibromatosis Type ◽

Type I ◽

Ras Proteins ◽

Wild Type ◽

Conserved Residues ◽

Mutant Proteins ◽

Quantitative Analyses ◽

Intrinsic Gtpase Activity

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.

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Characterization of Human Wild-Type and Mutant Argininosuccinate Synthetase Proteins Expressed in Bacterial Cells

Enzyme and Protein ◽

10.1159/000474998 ◽

1994 ◽

Vol 48 (5-6) ◽

pp. 251-264 ◽

Cited By ~ 7

Author(s):

Nazma Shaheen ◽

Keiko Kobayashi ◽

Terazono Hiroki ◽

Tomoko Fukushige ◽

Masahisa Horiuchi ◽

...

Keyword(s):

Bacterial Cells ◽

Argininosuccinate Synthetase ◽

Wild Type ◽

Human Wild Type

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Importance of Rhodospirillum rubrum H+-Pyrophosphatase under Low-Energy Conditions

Journal of Bacteriology ◽

10.1128/jb.186.19.6651-6655.2004 ◽

2004 ◽

Vol 186 (19) ◽

pp. 6651-6655 ◽

Cited By ~ 25

Author(s):

Rodolfo García-Contreras ◽

Heliodoro Celis ◽

Irma Romero

Keyword(s):

Alternative Energy ◽

Rhodospirillum Rubrum ◽

Physiological Role ◽

Low Energy ◽

Energy Conditions ◽

Wild Type ◽

Membrane Bound ◽

Potential Conditions

ABSTRACT The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.

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Cloning and characterization of Fc alpha Rb, a novel Fc alpha receptor (CD89) isoform expressed in eosinophils and neutrophils

Blood ◽

10.1182/blood.v88.11.4229.bloodjournal88114229 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4229-4238 ◽

Cited By ~ 29

Author(s):

TB van Dijk ◽

M Bracke ◽

E Caldenhoven ◽

JA Raaijmakers ◽

JW Lammers ◽

...

Keyword(s):

Amino Acids ◽

Phosphatidyl Inositol ◽

Surface Expression ◽

Cross Linking ◽

Wild Type ◽

Human Eosinophil ◽

Transmembrane Glycoprotein ◽

Membrane Bound ◽

Intracellular Region

The Fc receptor for IgA (Fc alpha R, CD89) is a transmembrane glycoprotein found on monocytes, macrophages, neutrophils, and eosinophils. Here we describe the characterization of a novel isoform of the Fc alpha R cloned from a human eosinophil cDNA library. This clone, Fc alpha Rb, lacks the exon encoding the transmembrane/intracellular region of wild type Fc alpha R, which is replaced by 23 new amino acids. Expression of Fc alpha Rb mRNA could be detected in eosinophils and neutrophils. IIA1.6 murine pro-B cells transfected with Fc alpha Rb cDNA secrete high levels of the protein, but also a substantial amount of Fc alpha Rb is expressed at the cell membrane. Membrane-bound Fc alpha Rb binds IgA-coated beads equally well as wild type Fc alpha R. Surface expression is not affected by phosphatidyl inositol phospholipase C, indicating that glycosyl phosphatidyl inositol-linkage of Fc alpha Rb is not likely. In IIA1.6 cells expressing Fc alpha Rb and FcR gamma, which is necessary for signal transduction by wild type Fc alpha R, no tyrosine phosphorylation or Ca(2+)-mobilization could be observed after receptor cross-linking. These results indicate that Fc alpha Rb is likely to have a different function than wild-type Fc alpha R receptor.

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Characterization of TRP-1 mRNA levels in dominant and recessive mutations at the mouse brown (b) locus.

Genetics ◽

10.1093/genetics/126.2.451 ◽

1990 ◽

Vol 126 (2) ◽

pp. 451-459

Author(s):

I J Jackson ◽

D Chambers ◽

E M Rinchik ◽

D C Bennett

Keyword(s):

Mrna Levels ◽

Wild Type ◽

Recessive Mutations ◽

Activation Of Transcription ◽

Membrane Bound ◽

The Common ◽

Low Levels ◽

The Relationship ◽

Tyrosinase Related Protein

Abstract The mouse brown locus encodes a putative membrane-bound metalloenzyme, tyrosinase-related protein-1 (TRP-1). We have examined the effect on mRNA expression of the locus of a number of mutant alleles. The common null mutant allele, brown, produces wild-type levels of TRP-1 mRNA, which is nonfunctional. Another recessive allele, cordovan-Harwell, has an intermediate, dark-brown phenotype and produces only very low levels of presumably normal TRP-1 mRNA. Two dominant alleles appear to act by killing the melanocyte in which they are expressed. One of them, Light, has normal size and amounts of TRP-1 mRNA. The other, White-based brown, produces no detectable TRP-1 mRNA. It has a gross DNA rearrangement at the 5' end, and we speculate that this results in activation of transcription of sequences not usually seen in melanocytes, and that this is toxic to the cell. The relationship between phenotype and molecular structure at the locus is discussed, and we draw some general principles applicable to other developmental genes.

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Mutant strains of Acinetobacter calcoaceticus possessing additional mandelate dehydrogenases. Identification and preliminary characterization of the enzymes

Biochemical Journal ◽

10.1042/bj2090379 ◽

1983 ◽

Vol 209 (2) ◽

pp. 379-386 ◽

Cited By ~ 12

Author(s):

C A Hills ◽

C A Fewson

Keyword(s):

Type Strain ◽

Chelating Agents ◽

Wild Type Strain ◽

Acinetobacter Calcoaceticus ◽

Wild Type ◽

Opposite Pattern ◽

Similar Dependence ◽

Mutant Strains ◽

Membrane Bound

Acinetobacter calcoaceticus wild-type strain N.C.I.B. 8250 can grow on only the L(+)-isomer of mandelate but mutant strains have been isolated that can grow on D(-)-mandelate. These mutants contain a novel D(-)-mandelate dehydrogenase in addition to the original L(+)-mandelate dehydrogenase. A second wild-type strain, EBF 65/65, shows the opposite pattern and can grow on D(-)-mandelate but not on L(+)-mandelate; mutants have been isolated that possess an L(+)-mandelate dehydrogenase in addition to the original D(-)-mandelate dehydrogenase and can thus grow on L(+)-mandelate. Both L(+)- and D(-)-mandelate dehydrogenases, whether originally present or evolved, are very similar in many respects: they are membrane-bound and NAD(P)+-independent; their activities have similar dependence on temperature and pH; they are inhibited by oxalate but not by several metal-chelating agents; they are stereospecific in their action and are inhibited by the opposite stereoisomers. D(-)-Mandelate dehydrogenase is much more susceptible than L(+)-mandelate dehydrogenase to inhibition by HgCl2 and p-chloromercuribenzoate and is much more heat-labile.

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