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1.
Phenylalanine hydroxylase gene: novel missense mutation in exon 7 causing severe phenylketonuria. 总被引:5,自引:0,他引:5
By direct sequence analysis of 94 mutant phenylalanine hydroxylase alleles using polymerase chain reaction-based techniques, we identified a C to T transition in exon 7 of the human phenylalanine hydroxylase gene that is associated with RFLP haplotypes 1 and 4. A leucine for proline substitution at position 281 can be predicted from the nucleotide sequence of the mutant codon. Expression analysis in cultured mammalian cells after site-directed mutagenesis proved that the base substitution is a disease causing gene lesion. Dot-blot hybridization analysis using allele-specific oligonucleotides revealed that 25% of all mutant haplotype 1 alleles in the German population bear this mutation. In addition, this mutation could be detected on one mutant haplotype 4 allele. The fact that this mutation is associated with only 25% of all mutant haplotype 1 alleles suggests that multiple mutations may be associated with this haplotype. The occurrence of several different mutations would be in agreement with the clinical heterogeneity observed in the group of patients whose PKU alleles belong to haplotype 1. 相似文献
2.
Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli. 总被引:9,自引:4,他引:5
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The sec/prl gene products catalyze the translocation of precursor proteins from the cytoplasm of Escherichia coli. Recessive, conditionally lethal mutant alleles of these genes (sec mutations) cause a generalized defect in protein secretion; dominant suppressor mutant alleles (prl mutations) restore export of precursor proteins with altered signal sequences. In prl strains, a precursor protein with a defective signal sequence can be selectively targeted to the suppressor gene product. When a precursor LacZ hybrid protein is used, the targeted prl protein is inactivated by the large, toxic hybrid molecule, a result termed suppressor-directed inactivation (SDI). Using SDI, two different secretion-related complexes can be generated: a pretranslocation complex that contains a hybrid protein with an unprocessed signal sequence, and a translocation complex in which the hybrid protein is jammed in transmembrane orientation with the signal sequence cleaved. Additional Sec proteins that are contained within, and thus sequestered by, each of these complexes can be identified when their functional levels are lowered using the conditional lethal sec mutations. Results of this genetic analysis suggest a multistep pathway for protein secretion in which the translocation machinery assembles on demand. 相似文献
3.
Sommers CM Martin NP Akal-Strader A Becker JM Naider F Dumont ME 《Biochemistry》2000,39(23):6898-6909
Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)?Ala(3)?lpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors. 相似文献
4.
Cultured skin fibroblasts from a patient with T-cell immune deficiency and an absence of purine nucleoside phosphorylase activity in red cells were assayed for their capacity to metabolize inosine and guanosine. The cultured fibroblasts were lacking activity of nucleoside phosphorylase and, compared to normal fibroblasts, could incorporate only 2% and 4% of 14C-inosine and 3H-guanosine, respectively, into acid precipitable material. Autoradiography visually confirmed the failure of the NP deficient cell line to incorporate the nucleosides into nuclear material. The physiological mechanism by which the deficiency of purine nucleoside phosphorylase causes T-cell dysfunction remains unclear. 相似文献
5.
Altered pathway routing in a class of Salmonella enterica serovar Typhimurium mutants defective in aminoimidazole ribonucleotide synthetase
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In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP. 相似文献
6.
Summary The genetic heterogeneity at the phenylalanine hydroxylase (PAH) locus was studied in 88 families including 93 of the 105 children with phenylketonuria (PKU) or hyperphenylalaninemia (HPA) detected through the Swedish neonatal screening program from 1966 to the end of 1986. Haplotypes based on eight restriction fragment length polymorphisms (RFLPs) at the PAH locus could be constructed for 132 normal and 136 mutant alleles. The normal alleles were of 27 different RFLP haplotypes, 9 of which have not been described previously, but there was a dominance of a few haplotypes common to many European populations. The distribution of mutant alleles was significantly different from that in neighboring countries, even though over 90% of all mutant alleles were confined to six RFLP haplotypes, also prevalent in other European populations. Allele-specific oligonucleotide hybridization analysis for the Arg408 to Trp408 mutation and for the G to A splicing mutation in intron 12 showed exceptions to the previously reported linkage of these mutations to mutant haplotypes 2 and 3, respectively. Correlation of mutant alleles with clinical phenotypes pointed to the presence of at least two different mutations associated with each of six haplotypes. We argue that PKU/HPA in the Swedish population may be caused by at least 13 different mutations in addition to the 4 already identified. The theoretical informativity of RFLP analysis in heterozygote detection and prenatal diagnosis in PKU/HPA families was estimated at approximately 85%. Carrier detection could, in effect, be accomplished for 88% of the 56 healthy siblings in the families studied. 相似文献
7.
8.
We report the molecular characterization of two novel galactosemia mutations that exhibit different molecular phenotypes. Both are of the missense type with low or no residual enzyme activity. The R148W mutation results in an unstable protein, although messenger RNA is still produced. In contrast, the L195P mutation produces stable but inactive immunoreactive protein. The R148W mutation alters an amino acid that is not evolutionarily conserved, while the L195P mutation affects a well-conserved residue nine amino acids down-stream from the putative active site nucleophile. These mutations provide evidence that different mechanisms can result in galactosemia: destabilizing mutations in any given area of the protein and missense mutations in conserved domains of the enzyme resulting in low or no activity. These two mutant alleles represent the fifth and sixth galactosemia mutations and confirm the hypothesis that galactosemia results from a multiplicity of mutations at the molecular level. 相似文献
9.
To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G(102) and G(112)) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site. 相似文献
10.
Multiple phenotypes associated with Myc-induced transformation of chick embryo fibroblasts can be dissociated by a basic region mutation.
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Chimaeric alleles were constructed to assay the biological functions of an N-terminal deletion and C-terminal mutations which were found in a naturally occurring mutant of feline vMyc, T17. The mutant alleles were assayed for their ability to transform chick embryo fibroblasts in vitro by a number of criteria, namely the ability to induce morphological transformation, an accelerated growth rate and growth in soft agar. Feline cMyc could transform the avian cells, whilst T17 vMyc could not, and the N-terminal deletion was responsible for conferring the primary transformation defect on the mutant protein. The C-terminal mutations which consist of a point mutation adjacent to the nuclear localisation signal and a point mutation/amino acid insertion within the basic region (BR) could, however, dissociate the Myc-induced parameters of transformation. This effect was a specific function of the BR mutation alone, and the mutation could be transferred into avian cMyc with comparable biological consequences. The BR mutation did not disrupt the sequence specific DNA binding activity of the protein in vivo, despite exerting a biological effect. These data suggest a novel phenotype where the mutation may affect a subset of Myc-regulated genes through altered DNA binding specificity or protein-protein interactions. 相似文献
11.
The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human
recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T
patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging
to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method.
As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant
in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a
loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5
in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant
alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A
substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common
haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant
among Japanese ATM mutant alleles.
Received: 15 September 1997 / Accepted: 12 January 1998 相似文献
12.
Single point mutations in domain II of the yeast mitochondrial release factor mRF-1 affect ribosome binding. 总被引:1,自引:0,他引:1
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We have recently described two yeast strains that are mutated in the MRF1 gene encoding the mitochondrial release factor mRF-1. Both mutants provoke gene-specific defects in mitochondrial translational termination. In the present study we report the cloning, sequencing, as well as an analysis of residual activities of both mutant mrf1 alleles. Each allele specifies a different single amino acid substitution located one amino acid apart. The amino acid changes do not affect the level or cellular localization of the mutant proteins, since equal amounts of wild type and mutant mRF-1 were detected in the mitochondrial compartment. Over-expression of the mutant alleles in wild type and mrf1 mutant yeast strains produces a phenotype consistent with a reduced affinity of the mutant release factors for the ribosome, indicating that the mutations map in a release factor domain involved in ribosome binding. We also demonstrate that nonsense suppression caused by a mutation in the mitochondrial homolog of the E. coli small ribosomal protein S4 can be reversed by a slight over-expression of the MRF1 gene. 相似文献
13.
14.
Genetic Evidence for Functional Interactions between Actin Noncomplementing (Anc) Gene Products and Actin Cytoskeletal Proteins in Saccharomyces Cerevisiae 总被引:7,自引:0,他引:7
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We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation. 相似文献
15.
16.
Venturi V Davenport MP Swan NG Doherty PC Kedzierska K 《Immunology and cell biology》2012,90(2):216-223
The emergence of the novel reassortant A(H1N1)-2009 influenza virus highlighted the threat to the global population posed by an influenza pandemic. Pre-existing CD8(+) T-cell immunity targeting conserved epitopes provides immune protection against newly emerging strains of influenza virus, when minimal antibody immunity exists. However, the occurrence of mutations within T-cell antigenic peptides that enable the virus to evade T-cell recognition constitutes a substantial issue for virus control and vaccine design. Recent evidence suggests that it might be feasible to elicit CD8(+) T-cell memory pools to common virus mutants by pre-emptive vaccination. However, there is a need for a greater understanding of CD8(+) T-cell immunity towards commonly emerging mutants. The present analysis focuses on novel and immunodominant, although of low pMHC-I avidity, CD8(+) T-cell responses directed at the mutant influenza D(b)NP(366) epitope, D(b)NPM6A, following different routes of infection. We used a C57BL/6J model of influenza to dissect the effectiveness of the natural intranasal (i.n.) versus intraperitoneal (i.p.) priming for generating functional CD8(+) T cells towards the D(b)NPM6A epitope. In contrast to comparable CD8(+) T-cell responses directed at the wild-type epitopes, D(b)NP(366) and D(b)PA(224), we found that the priming route greatly affected the numbers, cytokine profiles and TCR repertoire of the responding CD8(+) T cells directed at the D(b)NPM6A viral mutant. As the magnitude, polyfunctionality, and T-cell repertoire diversity are potential determinants of the protective efficacy of CD8(+) T-cell responses, our data have implications for the development of vaccines to combat virus mutants. 相似文献
17.
Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium 总被引:3,自引:1,他引:2
Johanna Björkman Patrik Samuelsson Dan I. Andersson & Diarmaid Hughes 《Molecular microbiology》1999,31(1):53-58
Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL , being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity ( ram ) phenotype. 相似文献
18.
Rodríguez-Pombo P Pérez-Cerdá C Pérez B Desviat LR Sánchez-Pulido L Ugarte M 《Biochimica et biophysica acta》2005,1740(3):489-498
Mutations in the PCCA or PCCB genes coding for alpha and beta subunits of propionyl CoA carboxylase can cause propionic acidemia. To understand the molecular basis of the intragenic complementation previously reported at the PCCB locus, we now examine the complementation behaviour of four carboxy-terminal and 11 amino-terminal naturally occurring mutant alleles both using cell fusion and reconstructing the complementation event by transfecting the mutant cDNAs to generate multimeric hybrid proteins. Alleles carrying mutations p.R410W and p.W531X are able to complement with 10 out of 11 amino-terminal mutations assayed. Only the unstable p.R512C, p.L519P and p.G112D mutants fail to complement. The results analyzed in the framework of the crystal structure of the homologous 12S transcarboxylase from Propionibacterium shermanii show that all mutant alleles studied are located at beta subunits interfaces, complementing alleles at the inter-trimer interface, where the catalysis probably happens, and non-complementing alleles at the intra-trimer interface, probably disrupting the trimer formation. Our results also show a remarkable stabilization effect when p.R410W is cotransfected with p.G246V. We propose a model for intragenic complementation requiring the production of two different beta subunits carrying carboxy and amino-terminal mutations that allow regenerating functional active sites and in which a stabilization effect between subunits could be relevant to ameliorate the biochemical phenotype of each mutation separately. 相似文献
19.
A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation. 总被引:10,自引:6,他引:4
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We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein. The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB. It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan. Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional. High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid. By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations. Many of the mutations decrease expression of the TrpC-TonB protein, as expected. In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB. The mutant proteins are designated TrpC-TonBG26D. The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations. TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm. We conclude that the Gly----Asp substitution is an export mutation. TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein. 相似文献
20.
The segment polarity gene costal-2 in Drosophila. I. The organization of both primary and secondary embryonic fields may be affected 总被引:4,自引:0,他引:4
A series of loss of function alleles at the costal-2 locus is described. Embryos mutant for lethal alleles that are derived from a mutant female germ line display polarity defects on the larval segments. A posterior part of the segmental denticle belt is missing and in its place is a mirror-image duplication of the anterior part including the segment boundary. Maternally rescued embryos are lethal but have normal morphology. Hypomorphic alleles escape to adults that display pattern duplications on the wings and halteres. Dominant gain of function alleles at the Costal-1 locus are also described and data are presented that argue that these are neomorphic and act in trans to impair functioning of costal-2. Some wild-type isoalleles of costal-2 are particularly sensitive to interference from Costal-1 mutations and different combinations of these alleles with Costal-1 can lead to embryos in which the primary embryonic field is disrupted (bicaudal phenotype) and adults with pattern duplications on the anterior compartment of most body segments. 相似文献