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PubMed1995

Mutation scan of the D1 dopamine receptor gene in 22 cases of bipolar I disorder.

In a previous study [Coon et al., Am J Hum Genet 52:1234-1249, 1993], we found suggestive evidence of linkage between manic-depressive illness (MDI) in eight multiplex pedigrees and D5S62, a DNA marker mapping to the telomeric region of 5q. As the D1 dopamine receptor gene (DRD1) maps to this region and as alterations in dopaminergic neurotransmission have been indirectly implicated in the pathogenesis of MDI, we directly searched for mutations in the coding region of the DRD1 gene in 22 unrelated cases of bipolar I (BPI) disorder derived from multiplex families. Using single strand conformation polymorphism (SSCP) analysis, we did not observe any abnormal SSCP variants in the BPI cases that differed from controls.

Adult↗
PubMed2004

Evidence for linkage on chromosome 4p16.1 in Type 1 diabetes Danish families and complete mutation scanning of the WFS1 (Wolframin) gene.

AIMS: To investigate whether the WFS1 gene, the gene for Wolfram syndrome, is a susceptibility gene for more common forms of diabetes in the Danish population. METHODS: One hundred and fifty-two Danish Type 1 diabetes mellitus sib-pair families were genotyped for two microsatellite markers situated within 5 cM of the WFS1 gene and analysed for linkage and association using the sib-TDT. The entire coding region, the 5'UTR and 3'UTR of the WFS1 gene, were screened for mutations by direct sequencing in 29 selected Type 1 diabetes patients. Four of the identified mutations were tested for linkage and association in 255 Danish Type 1 diabetes families (including 103 simplex families). RESULTS: Evidence for linkage to Type 1 diabetes was found as the second most frequent allele of the marker D4S394 were transmitted 137 times (T = 61%) and not transmitted 88 times to affected offspring (Puc = 0.0011). Twelve mutations were found in the coding region and three mutations in the 3'UTR. No evidence for linkage and association to Type 1 diabetes was found testing four of the identified amino acid substitutions. CONCLUSIONS: Evidence of linkage to Type 1 diabetes was observed in the Danish family collection. However, no evidence of linkage and association was observed for any of the analysed polymorphisms, suggesting that other variations must be responsible for the observed evidence of linkage in the region.

Age of Onset↗
PubMed2000

Mutational scanning of a hairpin loop in the tryptophan synthase beta-subunit implicated in allostery and substrate channeling.

The tryptophan synthases from Escherichia coli and Salmonella typhimurium are tetrameric enzymes, with an elongated TrpA.TrpB.TrpB.TrpA structure. Structural studies have identified residues 277-283 of TrpB as a potentially important region for the allosteric communication between the TrpA and TrpB subunits and for the transport of indole between their active sites through a hydrophobic tunnel. To explore the functional role of this region, we analyzed the effects of 19 single and double mutations in TrpB on the tryptophan synthase (TSase) and serine deaminase (SDase) activities of the TrpB2 dimer, either in the presence or in the absence of the TrpA subunit. The mutations of residues 273-283 could be divided into 4 classes. Mutations 1278A, F280G and M282A decreased the SDase and TSase activities of TrpB2 to similar extents. F280A decreased the SDase activity of TrpB2 more than its TSase activity, whereas the reverse was true for Y279L. F280A decreased the activation factor of TrpB2 by TrpA, whereas F280G increased it. The reaction steps and intramolecular contacts that could be affected by the mutations are described. The sequence 278-IYFGM-282, which is present in E. coli and S. typhimurium, is only found in 5 out of 42 organisms, whereas the sequence VLHGX is found in 21 organisms. Our results identified several mutations that could be used as structural probes to analyze precisely the roles of residues 278-282 and their evolution.

Allosteric Site↗
PubMed1998

A mutation scanning approach for the identification of hookworm species and analysis of population variation.

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR-SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.

Ancylostomatoidea↗
PubMed2006

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations.

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3' to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.

Amino Acid Substitution↗
PubMed2004

Mutation scanning of the androgen receptor gene in patients with psychiatric disorders reveals highly conserved variants in alcoholic and phobia patients.

Sex steroids exert potent effects on mood and mental state in humans. They may contribute to the risk of psychiatric disorders. To investigate this hypothesis, coding and splice junction sequences of the androgen receptor gene were scanned in genomic DNA samples to search for variants affecting protein structure and expression (VAPSEs). Ninety-six schizophrenics, along with pilot samples of patients with bipolar disorder, attention-deficit hyperactivity disorder, alcoholism and autism were analyzed with DOVAM-S, a robotically enhanced, optimized form of single-strand conformation polymorphism analysis. A total of 669 kb of genomic sequence was analyzed. Two VAPSEs were identified: R726L was found in one of 17 scanned alcoholics, and P516S, a novel VAPSE, was identified in one of three phobia patients. There were no length trends of the CAG triplets associated with schizophrenia. R726L and P516S occur at highly conserved amino acids. Further study is required to assess whether these VAPSEs contribute to the risk of alcoholism or phobia or other diseases.

Alcoholism↗
PubMed1998

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

Animals↗
PubMed1997

Analysis of oligonucleotide probe affinities using surface plasmon resonance: a means for mutational scanning.

A novel strategy for real-time analysis of oligonucleotide probe hybridization based on detection by surface plasmon resonance is described. The design of the analysis, exploiting the rapid dissociation kinetics of short oligonucleotides from their hybridization templates, allows monitoring in genuine sensor mode of equilibrium hybridization responses, circumventing the need for regeneration between sample cycles. Applied to a model system comprising oligonucleotide probes and different immobilized hybridization targets the effects of temperature, probe length, and nucleotide substitutions in template were investigated. The procedure described was observed to have an efficient discriminatory power with respect to end-mismatch situations. Affinity determinations of octamer probes showed good correlation between calculated Tm-values and probe affinities. From affinity data collected at different temperatures thermodynamic parameters were determined, which correlated well with data obtained from theoretical calculations. The technique, modified to a simplified form, allowed detection of single nucleotide substitutions in a target template, suggesting that procedures for confirmatory DNA sequencing can be envisioned.

Biotin↗
PubMed2003

Mutation scan of a type 1 diabetes candidate gene: the human interleukin-18 binding protein gene.

Type 1 (insulin-dependent) diabetes, T1DM, is the result of an immune-mediated destruction of the pancreatic beta cells dependent mainly on T helper cells and macrophages. Interleukin-18 (IL-18) is a proinflammatory cytokine produced mainly by macrophages. IL-18 is capable of inducing T lymphocyte synthesis of IFNgamma, thereby skewing the T helper response toward a T helper type 1 (Th1) profile. IL-18 binding protein (IL18BP) neutralizes IL-18 and leads to a reduced Th1 response. Polymorphisms in IL18BP may affect the activity of IL-18 and the magnitude of the Th1 response and may play a role in the pathogenesis of T1DM. The aim of the study was therefore to identify polymorphisms in IL18BP and to test these for association with T1DM. We evaluated the human IL18BP gene on chromosome 11q13 as a candidate susceptibility gene for T1DM and scanned the entire IL18BP (promoter, exons 1-6, and 3'UTR) for polymorphisms using single-strand conformational polymorphism analysis and direct sequencing. We identified a total of 11 polymorphisms, all having allele frequencies ranging between 0.05 and 0.10. Four were in the 5'UTR: -257G-->T, -78C-->T, -65G-->A, and -59A-->G. Three were in intron 3: IVS3+140A-->C, IVS4-147G-->T, and IVS4-59G-->T. The last four, 38*A-->T, 48*T-->A, 388*C-->G, and 440*_441*insG, were in the 3'UTR of IL18BP. However, none of these were frequent enough to permit association studies in T1DM and we conclude that IL18BP does not contribute to the overall genetic susceptibility to type 1 diabetes.

Base Sequence↗
PubMed2005

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network.

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.

DNA↗
PubMed2004

Mutation scanning analysis of Marteilia sydneyi populations from different geographical locations in eastern Australia.

Marteilia sydneyi (Paramyxea) is the causative agent of QX disease in oysters. In spite of the economic impact of this disease, its origin and the precise reason(s) for its apparent spread in Australian waters are not yet known. Given such knowledge gaps, investigating the population genetic structure(s) of M. sydneyi populations could provide insights into the epidemiology and ecology of the parasite and could assist in its prevention and control. In this study, single strand conformation polymorphism (SSCP)-based analysis of a region (195 bp) of the first internal transcribed spacer (ITS-1) of ribosomal DNA was employed to investigate genetic variation within and among five populations of M. sydneyi from oysters from five different locations in eastern Australia. The analysis showed the existence of a genetic variant of M. sydneyi common to the Great Sandy Strait, and the Richmond and Georges Rivers, as distinct from variants at the Pimpama and Clarence Rivers. Together with historical and other information relating to the QX disease outbreaks in eastern Australia, the molecular findings support the proposal that the parasite originated in the Great Sandy Strait and/or Richmond River and then extended southward along the coast. From a technical perspective, the study demonstrated the usefulness of SSCP as a tool to study the population genetics and epidemiology of M. sydneyi.

Animals↗
PubMed2001

Mutation scanning analysis of sequence heterogeneity in the second internal transcribed spacer (rDNA) within some members of the Hypodontus macropi (Nematoda: Strongyloidea) complex.

Single-strand conformation polymorphism analysis was employed to investigate sequence variation in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA within and among individuals representing three operational taxonomic units (OTUs) of Hypodontus macropi from different species of Australian macropodid marsupials. Of the 96 nematodes analysed, totals of 3 (OTU1 from Petrogale persephone), 10 (OTU2 from Macropus robustus) and 7 (OTU9 from Macropus rufus) representative individuals were selected for DNA sequencing to characterise and estimate the magnitude of nucleotide variation in the ITS-2. While no unequivocal nucleotide difference in the ITS-2 was detectable within OTU1, most sequence variation (3/44.7%) detected within OTU2 and OTU9 was related chiefly to dinucleotide (CA, TA, or a combination of both) differences. This microsatellite variability in some H. macropi OTUs suggests that the ITS-2 rDNA may be subjected to slippage events during DNA replication, resulting in short dinucleotide repeat tracts being dispersed throughout the ITS-2 lineages, or possibly transposition and/or crossing-over events. Nucleotide variation in the ITS-2 of individual OTUs was related to the proposed secondary structure for the precursor ribosomal RNAs. Most of the sequence heterogeneity or polymorphism within OTU2 and OTU9 occurred in loops or bulges of the predicted secondary structure, which appear not to be under functional constraint. The findings of this study have implications for investigating speciation events and population differentiation in nematodes at the molecular level.

Animals↗
PubMed2002

Mutational scanning and affinity cleavage analysis of UhpA-binding sites in the Escherichia coli uhpT promoter.

UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from -80 to -50 and the weak-binding (W) region from -50 to -32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (S)-1-(p-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.

Bacterial Proteins↗
PubMed2002

Complete mutation scan of the human Fas ligand gene: linkage studies in Type I diabetes mellitus families.

AIMS/HYPOTHESIS: Type I (insulin-dependent) diabetes mellitus is the result of a T-cell regulated selective destruction of pancreatic beta cells. There is evidence that the apoptosis inducing T-cell effector, Fas ligand (FasL) could be involved in the pathogenesis of Type I diabetes, probably because FasL-mediated apoptosis is important in maintaining peripheral self-tolerance and in down-regulating an immune response. We therefore evaluated the human FasL gene FASL on chromosome 1q23 as a candidate susceptibility gene for Type I diabetes. METHODS: The entire FASL (promoter, exons 1-4 and 3'UTR) was scanned for polymorphisms using single strand conformational polymorphism-heteroduplex analysis and direct sequencing. RESULTS: We identified two novel polymorphisms, a g-C843T and a g-A475T, in a negative regulatory region of the promoter. A Danish Type I diabetes family collection of 1143 subjects comprising 257 families (420 affected and 252 unaffected offspring) was typed for the g-C843T polymorphism and for a FASL microsatellite. Haplotypes were established and data were analysed using the extended transmission disequilibrium test. CONCLUSION/INTERPRETATION: We found no overall evidence for linkage in the presence of association of the FASL polymorphism to Type I diabetes and conclude that FASL does not contribute to the genetic susceptibility to Type I diabetes.

Amino Acid Substitution↗
PubMed1989

Construction of linker-scanning mutations using a kanamycin-resistance cassette with multiple symmetric restriction sites.

We demonstrate how a kanamycin-resistance (KmR) cassette flanked by polylinkers with multiple restriction sites can be used to introduce nucleotide (nt) sequence replacements into a region of interest. This method differs in two significant ways from traditional methods of linker mutagenesis. First, the presence of the KmR gene allows for selection of the polylinker, greatly facilitating formation of linker-containing molecules. Second, the polylinker with multiple restriction sites allows a given linker insertion to be combined with a second linker insertion in a variety of different ways and makes possible a range of novel nt to remain in the resulting linker replacement. The result of this flexibility is that fewer different molecules are needed to cover a region, and that relatively large replacements (greater than 40 nt) are possible. We have used this method to introduce a series of sequence replacements that span the mouse dihydrofolate reductase promoter region.

Animals↗
PubMed2005

Aspartic acid scanning mutation analysis of a goldfish growth hormone-releasing hormone (GHRH) receptor specific to the GHRHsalmon-like peptide.

Growth hormone-releasing hormone (GHRH) plays a pivotal role in the regulation of growth. The study of goldfish GHRH and its receptor is of particular interest as it is so far the only animal model in which two forms of GHRH-like (catfish-like and salmon-like) peptides coexist, and these peptides share only 30-40% of amino acid sequence identities with their mammalian counterparts. For these reasons, we have previously characterized a goldfish GHRH receptor, which is specific for a synthetic carp GHRH-like peptide. In this study, we investigated the structure-function relationships between the receptor and various ligands. Interestingly, among the two endogenous goldfish GHRH-like peptides, only the GHRHsalmon-like peptide was able to stimulate CHO cells transfected with the goldfish GHRH receptor. When the receptor was challenged by GHRHsalmon-like peptide either continuously for 45 min or periodically at 45-min intervals, mild homologous desensitization was observed. To determine whether the negatively charged residues of the receptor are responsible for discriminating GHRHsalmon-like from GHRHcatfish-like, 10 aspartic acid residues residing in the N-terminal ectodomain and the second exoloop were individually mutated to alanine by site-directed mutagenesis. Among these 10 mutants, four of them (D66A, D122A, D190A, and D196A) were defective as indicated by both cAMP assays and extracellular acidification rate measurements. Confocal microscopic studies showed that the D66A and D122A mutants, but not the D190A and D196A mutants, were expressed properly at the plasma membrane. Collectively, these results suggest that aspartic acid residues at positions 66 and 122 are critical for the interaction between the goldfish GHRH receptor and its endogenous ligands.

Animals↗
PubMed2001

Mutation scanning analysis of microsatellite variability in the second internal transcribed spacer (precursor ribosomal RNA) for three species of Metastrongylus (Strongylida: Metastrongyloidea).

This study investigated sequence variability in the second internal transcribed spacer of ribosomal DNA for 3 species of Metastrongylus (porcine lungworms). The ITS-2 region was amplified by PCR from individuals of M. elongatus, M. pudendotectus and M. salmi, and then subjected directly to single-strand conformation polymorphism analysis (SSCP), which allowed the direct display of sequence variation within and among individuals representing each species. There were marked differences in SSCP profiles among species, making this approach useful for species identification. For individual species, representative bands were excised from electrophoretic gels, reamplified by PCR and subjected to direct sequencing. For all 3 taxa, variability in the ITS-2 was related chiefly to the presence of microsatellites. Eight different microsatellites were identified, namely (A)n, (TG)n, (TCG)n, (TA)n, (TATG)n, (G)n, (TACA)n and (T)n. Considerable variability in microsatellite repeat number (ranging from 1 to 23) was found among individual nematodes of a species and between species. The microsatellites were located to specific stem or loop regions in the predicted ITS-2 precursor rRNA secondary structure. The results may suggest that slipped-strand mispairing in microsatellite regions contributes to sequence variability in the ITS-2 of Metastrongylus species under structural constraint as a consequence of microsatellite location in the precursor rRNA. Similar studies of the ITS-2 for a wide range of parasitic nematodes may lead to a better understanding of concerted evolution in these organisms.

Animals↗