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PubMed1994

A mutational analysis of receptor binding sites of interleukin-1 beta: differences in binding of human interleukin-1 beta muteins to human and mouse receptors.

The 3-D crystal structure of interleukin-1 beta (IL-1 beta) has been used to define its receptor binding surface by mutational analysis. The surface of IL-1 beta was probed by site-directed mutagenesis. A total of 27 different IL-1 beta muteins were constructed, purified and analyzed. Receptor binding measurements on mouse and human cell lines were performed to identify receptor affinities. IL-1 beta muteins with modified receptor affinity were evaluated for structural integrity by CD spectroscopy or X-ray crystallography. Changes in six surface loops, as well as in the C- and N-termini, yielded muteins with lower binding affinities. Two muteins with intact binding affinities showed 10- to 100-fold reduced biological activity. The surface region involved in receptor binding constitutes a discontinuous area of approximately 1000 A2 formed by discontinuous polypeptide chain stretches. Based on these results, a subdivision into two distinct local areas is proposed. Differences in receptor binding affinities for human and mouse receptors have been observed for some muteins, but not for wild-type IL-1 beta. This is the first time a difference in binding affinity of IL-1 beta muteins to human and mouse receptors has been demonstrated.

Amino Acid Sequence↗
PubMed2007

Alanine scanning mutational analysis of the ligand binding pocket of the human Vitamin D receptor.

We achieved exhaustive alanine scanning mutational analysis of the amino acid residues lining the ligand binding pocket of the Vitamin D receptor to investigate the mechanism of the ligand recognition by the receptor. This is the first exhaustive analysis in the nuclear receptor superfamily. Our results demonstrated the role and importance of all the residues lining the ligand binding pocket. In addition, this analysis was found to indicate ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the individual ligands. Thus, the analysis using 1beta-methyl-1alpha,25-dihydroxyvitamin D(3) revealed the specific van der Waals interactions of 1beta-methyl group with the receptor.

Alanine↗
PubMed2000

Mutation analysis of the p53 tumor suppressor gene using paraffin-embedded specimens of human transitional cell carcinomas by the direct sequencing method.

A very small quantity of DNA was extracted from paraffin sections of 25 bladder and 2 ureteral cancer cases, and exons 5-8 of the p53 gene were amplified by the polymerase chain reaction. Mutations were detected by direct sequencing, and their relationships to clinicopathological factors were assessed. Point mutations of the p53 gene were observed in 2 of 27 specimens of transitional cell carcinoma. One was a novel nonsense mutation. The prognosis of patients with p53 gene mutations was poorer than those of patients without mutations. Analysis of the data showed that assessment of the potential malignancy of bladder cancer, combined with a conventional pathological diagnosis, allows a more precise prognosis. Direct sequencing of tissue specimens enables swift prognostic evaluation and prompt decisions concerning treatment.

Adult↗
PubMed1999

Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis.

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.

Animals↗
PubMed2003

Mutation analysis of K-ras and beta-catenin genes related to O6-methylguanin-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma.

O6-methylguanin-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is the first step by alkylating agents in inducing DNA mutations in an organism. When MGMT and the mismatch repair (MMR) system are impaired, O6-methylguanine mispairs with thymine during DNA replication, resulting in a G:C right curved arrow A:T transitional mutation in DNA. We obtained cancer lesions by manual micro-dissection (MMD) from 26 paraffin-embedded formalin-fixed gallbladder carcinoma and Laser Capture Micro-dissection (LCM) method from 10 fresh frozen specimens. Mutation analysis was performed on the micro-dissected samples for K-ras and beta-catenin genes. At codon 12 of the K-ras gene, the MMD and LCM methods detected mutations in 3 (11.5%) and 1 (10%) case, respectively. In exon 3 of beta-catenin gene, only 1 (3.8%) case revealed a mutation in MMD cancer foci. Two cases without MGMT or MMR expression revealed a G right curved arrow A transition mutation in the K-ras gene. The findings suggested that negative MGMT and MMR status contributed to a G:C right curved arrow A:T transitional mutation in the K-ras gene. However, K-ras and beta-catenin mutations were actually rare in GB carcinoma. Other gene mutations frequently occurring in gallbladder carcinoma might be affected by this negative MGMT and MMR status.

Base Pair Mismatch↗
PubMed1995

Genomic DNA organization of human mitochondrial very-long-chain acyl-CoA dehydrogenase and mutation analysis.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a major enzyme catalyzing long-chain fatty acids in the first step of mitochondrial beta-oxidation system. Inborn error of this enzyme can cause sudden infant death syndrome and hypertrophic cardiomyopathy is present at a significantly high frequency. To investigate VLCAD deficiency at the genomic DNA level, we cloned the VLCAD gene and analyzed the structure. The gene is about 5.4 kb long and contains 20 exons. We performed mutation analysis in two patients, both having a 105 bp deletion encompassing bases 1078-1182 in cDNA. A point mutation (GT-->AT) at 5' splice site of intron 11 was identified in both patients. This mutation seems to cause skipping of exon 11 corresponding to the 105 bp deletion. This is the first documentation of aberrant splicing in the VLCAD gene.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed2007

Mutational analysis of NPHS2 and WT1 in frequently relapsing and steroid-dependent nephrotic syndrome.

Idiopathic nephrotic syndrome is a common pediatric kidney disease, 80% of all cases are steroid sensitive (SSNS). A significant proportion of children with SSNS will have a frequently relapsing or steroid-dependent course (FRNS/SDNS) that is associated with significant treatment-related morbidity. Mutations in NPHS2 account for more than 28% of all cases of steroid-resistant nephrotic syndrome (SRNS) and dominant mutations in WT1 for 5%; while mutations are absent from children with uncomplicated SSNS. Since FRNS/SDNS is phenotypically positioned within a spectrum between SSNS and SRNS, we hypothesized that heterozygous mutations of NPHS2 may be causing FRNS/SDNS. Mutational analysis of NPHS2 and WT1 was carried out in a single-center cohort of 20 children with FRNS/SDNS, ten children with uncomplicated SSNS (control), and 22 children with SRNS (control). Renal biopsy findings were available in 15/20 children with FRNS/SDNS and revealed IgM nephropathy, MCNS, and FSGS in six, five, and four children, respectively. Children with FRNS/SDNS were significantly younger at first presentation than those with SSNS and SRNS (median age: 3.0 years in FRNS/SDNS patients, 7.0 years in SSNS patients, and 5.0 in SRNS patients; p < 0.001). No NPHS2 or WT1 mutations were found in patients with FRNS/SDNS and uncomplicated SSNS. The hypothesis that FRNS/SDNS may be associated with heterozygous mutations in NPHS2 or WT1 was not confirmed.

Adolescent↗
PubMed2006

Reverse mutational analysis reveals threonine-373 as a potentially sufficient phosphorylation site for inactivation of the retinoblastoma tumor suppressor protein (pRB).

Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin D-CDK activity in the inactivation of the retinoblastoma protein (pRb). Rb(DeltaCDK), a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E overexpression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a "reverse mutational analysis" by restoring cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to Rb(DeltaCDK), alone or in combination with other phospho-restorations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by Rb(DeltaCDK), but not by mutants of Rb(DeltaCDK) containing a restored threonine-373. Finally, while S phase entry is blocked by expression of Rb(DeltaCDK), restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.

Animals↗
PubMed1995

Mutational analysis of Rox1, a DNA-bending repressor of hypoxic genes in Saccharomyces cerevisiae.

Rox1 is a repressor of the hypoxic genes of Saccharomyces cerevisiae. It binds to a specific hypoxic consensus sequence in the upstream region of these genes and represses transcription in conjunction with the general repression complex Tup1-Ssn6. In this study, we demonstrated that the first 100 amino acids comprising the HMG domain of Rox1 were responsible for DNA binding and that when bound, Rox1 bent DNA at an angle of 90 degrees. A mutational analysis resulted in the isolation of seven missense mutations, all located within the HMG domain, that caused loss of DNA binding. The effect of these mutations on the structure of Rox1 was evaluated on the basis of the homology between Rox1 and the human male sex-determining protein SRY, for which a structural model is available. The failure to isolate missense mutations in the carboxy-terminal three-quarters of the protein prompted a deletion analysis of this region. The results suggested that this region was responsible for the repression function of Rox1 and that the repression information was redundant. This hypothesis was confirmed by using a set of fusions between sequences encoding the GAL4 DNA-binding domain and portions of ROX1. Those fusions containing either the entire carboxy-terminal region or either half of it were capable of repression. Repression by selected fusions was demonstrated to be dependent on Ssn6.

Base Sequence↗
PubMed2000

Mutational analysis of RET/GDNF/NTN genes in children with total colonic aganglionosis with small bowel involvement.

Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the distal gut, resulting in bowel obstruction shortly after birth. Aganglionosis usually affects the distal colon, but may also extensively involve the entire colon and, rarely, the more proximal bowel. Recently, germline mutations of RET, GDNF, and NTN genes have been reported in HSCR. Here we describe the results of mutational analysis of these genes in 15 Japanese child patients with total colonic aganglionosis with small bowel involvement. DNA sequences of all the RET/GDNF/NTN coding regions were determined by the direct dyedeoxy terminator cycle method. Eight different RET mutations were identified in exons 1, 7, 10, 12, 15, and 17 in 10 of the 15 patients. Of these eight mutations, five were found in the tyrosine kinase domain. No GDNF or NTN mutation was found. Compared with typical HSCR, this patient group appeared to exhibit a higher percentage of RET mutations and accumulation of mutations in the tyrosine kinase domain. A homozygous (or hemizygous) RET mutation was found in a male baby with total intestinal aganglionosis, while the heterozygosity of the same mutation resulted in a less severe type of aganglionosis. In familial cases, all heterozygous for the same mutation, aganglionosis was more severe in male than in female siblings. These results also urge us to examine if the RET germline mutation may cause critical alteration of the GDNF/NTN-Ret signal transduction more severely in homo(hemi)zygosity and in male fetuses during organogenesis.

Child↗
PubMed1994

Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli.

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.

Bacteriophage lambda↗
PubMed2001

Functional and mutational analysis of p19, a DNA transfer protein with muramidase activity.

Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems. We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants. The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17. Overexpression of protein P19 in an early growth phase resulted in a massive lysis of Escherichia coli cells in liquid culture, as indicated by a rapid and distinct decrease in cell culture densities after induction. Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect. P19-induced cell lysis was directly shown by transmission and scanning electron microscopy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall. To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family. From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity. This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.

Amino Acid Sequence↗
PubMed2002

Mutational analysis of the discs large tumour suppressor identifies domains responsible for human papillomavirus type 18 E6-mediated degradation.

The discs large (Dlg) tumour suppressor protein is targeted for ubiquitin-mediated degradation by the high-risk human papillomavirus E6 proteins. To understand further the mechanisms behind this, a mutational analysis of Dlg was undertaken. This study demonstrates that an intact PDZ domain 2 (PDZ2) on Dlg is necessary for the ability of E6 to bind and degrade Dlg. However, additional residues within the amino-terminal portion of Dlg are also required for optimal E6 activity. Stable cell lines expressing different Dlg mutants were also established and these confirm that Dlg is regulated intrinsically by the proteasome in the absence of E6; however, in this case, the sequences responsible for regulating Dlg stability lie predominantly within PDZ2. These results suggest that there are at least two mechanisms for regulating Dlg protein stability and that the pathways used by E6 are not necessarily the same as those used in the cell in its absence.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Mutational analysis of ETV6 in prostate carcinoma.

BACKGROUND: In an effort to better understand the molecular events responsible for progression of prostate carcinoma to metastatic disease, we have recently identified a homozygous deletion at 12p12-13 involving ETV6 (tel). Although mutational analysis of ETV6 has not been examined previously in prostate carcinoma, it is an attractive candidate prostate cancer tumor suppressor gene since as it previously has been implicated in malignancy. Therefore, we decided to analyze 43 prostate cell lines, xenografts, and metastatic foci for inactivating mutations. METHODS: DNA was isolated from 7 cell lines, 18 xenografts, and 18 metastatic deposits. Single-strand conformational polymorphism (SSCP) analysis of ETV6, was performed by polymerase chain reaction (PCR) amplification of each exon by using intron specific primers. PCR products were then resolved by gel electrophoresis, and aberrantly migrating PCR products were then sequenced. RESULTS: Two previously described polymorphisms and four novel sequence changes were identified. Polymorphisms at nucleotide 258 (G --> A, Thr --> Thr) and 602 (T --> C, Leu --> Pro) were identified in eight and one specimen(s), respectively. Analysis of noncancerous DNA confirmed the presence of the polymorphisms in the germ-line. Four possible mutations were identified at nucleotides 24 (T --> G, Cys --> Trp), 380 (G --> A, Arg --> Glu), 776 (G --> T, Arg --> Leu), and 876 (C --> T, Leu --> Leu). Three were in xenografts or cell lines. Because normal DNA was not available, these could represent rare polymorphisms. The sole mutation in a clinical specimen, at nucleotide 876, did not result in an amino acid change. CONCLUSION: Our data suggest that mutational inactivation ETV6 may occur in prostate carcinoma. The functional significance of these potential inactivating mutations remains to be determined.

Carcinoma↗
PubMed2005

Mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in Peutz-Jeghers syndrome.

Mutations in LKB1 lead to Peutz-Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in 28 LKB1-negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Global transcriptome analysis of the cold shock response of Shewanella oneidensis MR-1 and mutational analysis of its classical cold shock proteins.

This study presents a global transcriptional analysis of the cold shock response of Shewanella oneidensis MR-1 after a temperature downshift from 30 degrees C to 8 or 15 degrees C based on time series microarray experiments. More than 700 genes were found to be significantly affected (P < or = 0.05) upon cold shock challenge, especially at 8 degrees C. The temporal gene expression patterns of the classical cold shock genes varied, and only some of them, most notably so1648 and so2787, were differentially regulated in response to a temperature downshift. The global response of S. oneidensis to cold shock was also characterized by the up-regulation of genes encoding membrane proteins, DNA metabolism and translation apparatus components, metabolic proteins, regulatory proteins, and hypothetical proteins. Most of the metabolic proteins affected are involved in catalytic processes that generate NADH or NADPH. Mutational analyses confirmed that the small cold shock proteins, So1648 and So2787, are involved in the cold shock response of S. oneidensis. The analyses also indicated that So1648 may function only at very low temperatures.

Adaptation, Physiological↗
PubMed2000

Mutational analysis of two putative catalytic motifs of the type IV restriction endonuclease Eco57I.

The role of two sequence motifs (SM) as putative cleavage catalytic centers (77)PDX(13)EAK (SM I) and (811)PDX(20)DQK (SM II) of type IV restriction endonuclease Eco57I was studied by site-directed mutational analysis. Substitutions within SM I; D78N, D78A, D78K, and E92Q reduced cleavage activity of Eco57I to a level undetectable both in vivo and in vitro. Residual endonucleolytic activity of the E92Q mutant was detected only when the Mg(2+) in the standard reaction mixture was replaced with Mn(2+). The mutants D78N and E92Q retained the ability to interact with DNA specifically. The mutants also retained DNA methylation activity of Eco57I. The properties of the SM I mutants indicate that Asp(78) and Glu(92) residues are essential for cleavage activity of the Eco57I, suggesting that the sequence motif (77)PDX(13)EAK represents the cleavage active site of this endonuclease. Eco57I mutants containing single amino acid substitutions within SM II (D812A, D833N, D833A) revealed only a small or moderate decrease of cleavage activity as compared with wild-type Eco57I, indicating that the SM II motif does not represent the catalytic center of Eco57I. The results, taken together, allow us to conclude that the Eco57I restriction endonuclease has one catalytic center for cleavage of DNA.

Amino Acid Motifs↗
PubMed2006

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene.

The identification of mutations in the MEN1 gene causing MEN1 has represented a challenge since the cloning of the gene in 1997 because of the lack of mutation hot-spots in the gene and the lack of phenotype-genotype correlations. The use of denaturing high performance liquid chromatography (DHPLC), a high throughput, reliable and automated heteroduplex-based technique, is the ideal for mutation detection in MEN1. In this work, DHPLC was optimised for the screening of the nine coding exons and splice junctions of MEN1. Thanks to collaboration between two French laboratories recognised as reference centres for genotypic MEN1 diagnosis (Lyon and Lille), a blind retrospective study conducted in a cohort of 160 unrelated MEN1 probands with (or without) known germline mutations was undertaken to evaluate the sensitivity of DHPLC. We were able to detect 101 different sequence variations by DHPLC, distributed in the 10 analysed DNA fragments and corresponding to 100% of mutation detection compared with direct sequencing. 1.2% of samples were considered as false positive, exhibiting a heterogenous profile. DHPLC did not detect five cases of deletion or duplication of complete exons, neither did direct sequencing, showing the limits of the technique. Nevertheless, the method appeared to allow automated, rapid and low-cost mutation detection with high accuracy. Direct sequencing can be then applied to identify the sequence variations on the targeted DNA fragments showing heterozygous profile by DHPLC. In conclusion, genotypic diagnosis of MEN1 can benefit from DHPLC in terms of efficacy, rapidity and cost.

Base Sequence↗