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At least 1,567 records · Page 87Linked to original sources
PubMed2005

Mutational analysis of target enzyme recognition of the beta-trefoil fold barley alpha-amylase/subtilisin inhibitor.

The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k(on), whereas the k(off) values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K(i) values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca(2+) at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca(2+) had weaker affinity for AMY2 and reduced sensitivity to the Ca(2+) modulation of the affinity. These results suggest that the Ca(2+) and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases.

Amino Acid Sequence↗
PubMed2006

Mutation analysis of the seven in absentia homolog 1 (SIAH1) gene in Parkinson's disease.

Seven in absentia homolog 1 (SIAH-1) is a member of the RING-finger-containing E3 ubiquitin ligases. Two substrates of SIAH-1 are alpha-synuclein and synphilin-1, both of these proteins are involved in Parkinson's disease (PD). Recently, mutations in Parkin, another E3 ubiquitin ligase which ubiquinates synphilin-1 and glycosylated alpha-synuclein, have been defined as a major cause of autosomal recessive PD. The potential role of SIAH-1 in PD is further underlined as SIAH-1 protein is a component of the Lewy bodies and as it plays a role in apoptosis caused by nitric oxide (NO) induced oxidative stress. Thus, we performed a mutation screening of the SIAH-1 gene in PD patients. However, screening a large sample of 209 familial and sporadic PD patients we could not find any disease causing mutation. We therefore conclude that genetic alterations of SIAH-1 do not significantly contribute to the pathogenesis of PD.

Aged↗
PubMed1999

Systematic mutation analysis of the catechol O-methyltransferase gene as a candidate gene for schizophrenia.

OBJECTIVE: Catechol O-methyltransferase (COMT) is involved in the degradation of catecholamine neurotransmitters. Recent linkage studies of schizophrenia and molecular studies of velocardiofacial syndrome suggest that the COMT gene might be a candidate gene for schizophrenia. METHOD: The authors systematically searched for mutations and microdeletion of the COMT gene in 177 Chinese schizophrenic patients from Taiwan; 99 comparison subjects were also studied. RESULTS: Five molecular variants were identified: c.186C > T at exon 3, c.408C > G at exon 4, c.472G > A at exon 4, c.597G > A at exon 5, and c.821-827insC at the 3' untranslated region. However, no differences in the genotype and haplotype frequencies of these molecular variants between the schizophrenic and comparison subjects were detected. Furthermore, no microdeletion was identified among the patients. CONCLUSIONS: These data suggest that the COMT gene does not play a major role in the pathogenesis of schizophrenia, and the genotypic overlap between schizophrenia and velocardiofacial syndrome was rare in this cohort.

Catechol O-Methyltransferase↗
PubMed1997

Mutational analysis of the rolA gene of Agrobacterium rhizogenes in tobacco: function of the rolA pre-mRNA intron and rolA proteins defective in their biological activity.

The rolA gene of Agrobacterium rhizogenes contains in its untranslated leader region a spliceosomal intron, which is spliced in Arabidopsis and in Nicotiana tabacum. Expression under the control of the 35S promoter from cauliflower mosaic virus of a rolA gene derivative defective in splicing still causes alterations of growth in transgenic tobacco plants. Splicing of rolA mRNA is required for efficient expression of the rolA phenotype in vivo. Moreover, splicing is required for efficient in vitro translation of the rolA mRNA. In contrast, expression of a 35S-rolA gene derivative with the ATG initiation codon replaced by ATA does not cause any phenotypical alteration. Mutations leading to amino acid substitutions at positions 37 and 40 of the rolA coding region were isolated as null mutants in Arabidopsis plants transgenic for the rolA gene. However, when expressed in tobacco under the control of the 35S promoter, they cause a rolA phenotype reduced in the expressivity of its traits. The molecular characterization of rolA mutants might be useful for understanding the biochemical function of the rolA protein.

Bacterial Proteins↗
PubMed1998

Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.

An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.

Amino Acid Sequence↗
PubMed2003

Mutational analysis of the androgen receptor using laser capture microdissection and direct sequencing.

Molecular analysis of prostate cancer specimens is complicated by tumor heterogeneity and admixture of malignant cells with benign epithelium, stroma and inflammatory cells. Laser Capture Microdissection allows procurement of selected groups of cells from specific areas of tissue sections. We have used Laser Capture Microdissection to obtain pure samples of malignant prostate epithelial cells from frozen radical prostatectomy specimens. Methods for DNA extraction, androgen receptor amplification and exon sequencing have been optimized. These methods should be adaptable for molecular analysis of DNA, RNA and protein from other complex tissues.

DNA Mutational Analysis↗
PubMed2000

Impaired BAX protein expression in breast cancer: mutational analysis of the BAX and the p53 gene.

We have previously shown that the pro-apoptotic BAX protein is differentially expressed in breast cancer and in other epithelial tumors. In this line, a reduced BAX protein expression is a negative prognostic factor in various carcinomas including breast cancer. For p53, a trancriptional activator of BAX in apoptosis, mutations in the coding sequence were shown to modulate BAX protein expression in cell line models on the transcriptional level. We therefore investigated the BAX gene in 68 breast cancer specimens for the presence of mutations in the coding sequence by single-strand conformation polymorphism (SSCP)-PCR and direct sequencing. The expression of BAX protein was assessed by immunohistochemistry. In addition, we screened for mutations in the exons 5-8 of the p53 gene by SSCP-PCR to assess whether mutations in the DNA-binding domain of this upstream regulator of BAX gene transcription are responsible for differences in BAX protein expression. As previously observed, BAX was differentially expressed in the breast cancer samples, but no mutations in the coding sequence of the BAX gene were found besides a polymorphism in exon 6 at the position 552 (G->A) and additional intronic polymorphisms. In contrast, we identified 16 of 68 (23.5%) tumors to bear mutations in the p53 gene. In the subset of BAX-expressing tumors, the mutational inactivation of p53 did result in a reduced BAX protein expression (Fisher exact test, p = 0. 047). Nevertheless, we identified a subset of BAX-negative tumors lacking BAX or p53 mutations. Thus, additional, not yet identified regulators, apart from p53, appear to be involved in the regulation of BAX protein expression.

Base Sequence↗
PubMed2003

Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes.

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.

3T3-L1 Cells↗
PubMed2001

Mutational analysis of the high-affinity BvgA binding site in the fha promoter of Bordetella pertussis.

In order to define a consensus binding sequence for the response regulator BvgA, we have undertaken a systematic analysis of contributions made by each nucleotide within the heptad half-sites that are present in an inverted orientation at the promoter for the fha operon. Using in vitro binding assays, we examined the full complement of 21 single point mutations symmetrically arranged in this heptad repeat. Both gel shift and nitrocellulose filter-binding assays provided evidence that nucleotides at positions 3 (thymidine), 4 (cytosine) and 7 (adenine) in the binding heptad contribute substantially to sequence-specific recognition by BvgA. Furthermore, a T to A conversion at position 6 reduced binding. Selected binding site mutations were introduced into a modified fha promoter and examined for their effects on BvgA activation of promoter activity in vivo. Only those substitutions most severely affecting binding in vitro affected promoter activity in vivo. The in vivo effects of substitutions that had a significant effect on binding in vitro but did not severely affect in vivo promoter activity under standard culture conditions could be detected in vivo either in combination with additional substitutions or from their effect on the sensitivity of the mutant promoters to modulation by magnesium sulphate.

Bacterial Outer Membrane Proteins↗
PubMed1998

Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1.

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopus oocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.

Animals↗
PubMed2002

Refinement of the genomic structure of STX1A and mutation analysis in nondeletion Williams syndrome patients.

Williams syndrome (WS) is a contiguous gene deletion disorder in which the commonly deleted region contains at least 17 genes. One of these genes, Syntaxin 1A (STX1A), codes for a protein that is highly expressed in the nervous system and is essential for the docking of synaptic vesicles with the presynaptic plasma membrane. In this study, we refine the complete genomic structure of the human STX1A gene by direct sequencing and primer walking of bacterial artificial chromosome (BAC) clones and show that STX1A contains at least 10 exons and 9 introns. The length of exons range from 27 bp to 138 bp and all splice sites conform to the GT-AG rule. Investigation of the STX1A gene sequence in five WS patients without detectable deletions did not identify any point mutations. Although the regulatory elements that control STX1A transcription were not examined, these results do not support a role for STX1A in the WS phenotype.

Adolescent↗
PubMed2004

Mutation analysis of the KAL gene in female patients with gonadotropin-releasing hormone deficiency.

Isolated gonadotropin-releasing hormone (GnRH) deficiency, including Kallmann's syndrome (KS) and idiopathic hypogonadotropic hypogonadism (IHH), is a congenital disorder, which is characterized by a functional deficit in hypothalamic GnRH secretion. Despite recent advances in the understanding of the pathogenesis of the X-linked form of KS as the identification of the KAL gene (Xp22.3), the genetic basis of the sporadic form in female patients remains unclear. Although most searches for mutations in X chromosome have been reported in males, the newly recognized phenomenon of inheritance, such as genomic imprinting and uniparental disomy, raises the possibility of a female phenotype in the X- linked genetic defect. Here, the molecular study of the coding region of the KAL gene (exon 5 to 14) in 10 unrelated females with KS (n=6) or IHH (n=4) is reported. None of the subjects had familial histories of delayed puberty or hypogonadism. Samples from 4 healthy, unrelated female volunteers were used for identification of polymorphisms. PCR of the 10 exons of the KAL gene was performed on genomic DNA. The PCR products of the 10 exons were subject to single strand conformation polymorphism (SSCP) analysis to identify possible mutations. In an SSCP analysis of the amplified fragments (fragment size: 147 to 302 bp), no mutations or polymorphisms were found in any of the 10 patients and 4 controls. In conclusion, it is unlikely that KAL gene mutations are a clinically significant cause of sporadic GnRH deficiency in female patients, indicating the existence of defects in unidentified genes that result in the expression of the phenotypes in females.

Adolescent↗
PubMed2005

Mutational analysis of proapoptotic ARTS P-loop domain in common human cancers.

Mounting evidence indicates that deregulation of apoptosis is involved in mechanisms of cancer development. ARTS is released from mitochondria into the cytosol during apoptosis, promoting caspase activation by neutralizing the inhibition of inhibitor of apoptotic protein (IAP) on caspases. ARTS contains a P-loop GTP-binding domain, which is essential for the apoptotic function of ARTS. The aim of this study was to ascertain whether a genetic alteration of ARTS gene is involved in the development of human cancers possibly by inactivating the apoptotic function of ART. We analyzed the coding region of the P-loop domain of human ARTS gene for the detection of somatic mutations in 100 gastric carcinomas, 100 non-small cell lung cancers, and 69 hepatocellular carcinomas using a polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP). However, there was no mutation in the P-loop coding region in the cancers. The data presented here suggest that ARTS P-loop is not frequently mutated in gastric, lung, and hepatocellular carcinomas, and that apoptosis deregulation in cancers is not dependent on the mutation of ARTS gene.

Apoptosis↗
PubMed1994

Mutational analysis of residue 190 of human immunodeficiency virus type 1 reverse transcriptase.

S-2720 and other members of the quinoline/quinoxaline class of HIV-1-specific nonnucleoside reverse transcriptase inhibitors (NNRTIs) select for a glycine to glutamate substitution at residue 190 (Gly 190 Glu) of the reverse transcriptase (RT), when drug-resistant viruses are generated in cell culture. This mutation has not been described to appear upon selection for resistant viral variants using derivatives of any other class of NNRTIs. Notably, the RNA-dependent DNA polymerase activity of the Gly 190 Glu mutant enzyme is drastically diminished with respect to the wild-type RT. We describe here the effects of other amino acid substitutions at position 190 of the RT that were introduced by using site-directed mutagenesis. Polymerase activities and sensitivities to inhibition by a number of NNRTIs were determined for the different RT mutants. In general, an inverse correlation was found between the enzymatic activity and increasing length of the side chain, whereas the size of the residue and the level of resistance to NNRTIs appeared to be positively related. Double mutants, which contain the Gly 190 Glu mutation together with substitutions that confer resistance to other RT inhibitors, were all shown to possess severely diminished polymerase activity.

Antiviral Agents↗
PubMed1997

A mutational analysis of conjugation in Tetrahymena thermophila. 2. Phenotypes affecting middle and late development: third prezygotic nuclear division, pronuclear exchange, pronuclear fusion, and postzygotic development.

Conjugation following pair formation in Tetrahymena can be divided into three distinct sequences of events: prezygotic development, postzygotic development, and exconjugant development. The decision to proceed with postzygotic development is governed by a developmental checkpoint occurring sometime during the middle stages of conjugation. A second developmental decision is made to initiate pair separation and exconjugant development. This paper examines the phenotypes of five newly isolated conjugation mutants (cnj6-cnj10) which affect middle and late events within the conjugation program. cnj6 mutants exhibit normal nuclear behavior throughout development up to and including differentiation of new macronuclear anlagen. Pairs arrest at this developmental endpoint, unable to dissociate. cnj7 and cnj8 eliminate the third prezygotic nuclear division and the first postzygotic nuclear division. All subsequent developmental events appear normal. cnj9 eliminates the second postzygotic nuclear division, and subsequently, new macronuclei fail to develop despite parental macronuclear degradation. cnj10 results in a pleiotropic phenotype characterized by failure of numerous events which all appear to involve nuclear-cytoskeletal interactions. These defects include nuclear selection (anchoring nuclei to the exchange junction), pronuclear exchange, pronuclear fusion, and anchoring postzygotic nuclear division products to the posterior cell cortex. These mutant phenotypes are used to draw inferences regarding developmental dependencies that govern a cell's entry into the postzygotic and exconjugant developmental programs.

Animals↗
PubMed2003

Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure.

In eukaryotic DNA replication, replication factor C (RFC) acts as a "clamp loader" that loads PCNA onto a primed DNA template in an ATP-dependent manner. Proteins with functions essentially identical to that of RFC exist in Archaea. We have determined the crystal structure of the small subunit (RFCS) of Pyrococcus furiosus RFC at 2.8-A resolution. Using the information from the determined tertiary structure, we prepared several mutations in RFCS and biochemically characterized them. Truncation of the C-terminal alpha-helix (alpha16) causes a failure in RFCS oligomerization and a loss of the stimulating activity for the PCNA-dependent DNA synthesis by DNA polymerases. The site-directed reduction of the negative charges at the center part of the RFCS complex affected the stability of the RFC-PCNA interaction and reduced the clamp-loading activity. These results contribute to our general understanding of the structure-function relationship of the RFC molecule for the clamp-loading event.

Adenosine Triphosphatases↗
PubMed1989

The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements.

The activity and cell-type specificity of the promoter of the MF alpha 1 gene of Saccharomyces cerevisiae were examined by measuring expression of an MF alpha 1-SUC2 gene fusion in MATa, MAT alpha, and MATa/MAT alpha cells. A high level of invertase activity was observed only in MAT alpha cells. Weak expression occurred in MATa cells when the hybrid gene was carried on a multicopy plasmid or on a centromere-containing plasmid, but not when the hybrid gene was integrated at the normal MF alpha 1 locus. Analysis of a set of 5'-deletions of the promoter region of the MF alpha 1-SUC2 gene on the multicopy plasmid indicated that sequences from -354 to -274 upstream of the translational start site were required for high level expression in MAT alpha cells. Smaller internal deletions and insertions within the promoter region of the MF alpha 1-SUC2 gene were inserted into the genome at the normal MF alpha 1 locus. These mutations further delineated four promoter domains important for expression: (1) two 26 bp elements (-365 to -340 and -312 to -287) with imperfect dyad symmetry; (2) a 40 bp segment (-264 to -226) that lies about 120 bp upstream of the TATA box; and (3) the TATA box itself (-128 to -122). The transcriptional start sites of the normal MF alpha 1 promoter and of a mutant lacking the TATA box were determined. The MF alpha 1 locus was mapped to the left arm of chromosome XVI, about 22 cM centromere-proximal to the PEP4 gene.

Base Sequence↗