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PubMed2001

Single-strand conformation polymorphism analysis of genetic variation in Labiostrongylus longispicularis from kangaroos.

Single-strand conformation polymorphism (SSCP) analysis was employed to screen for sequence heterogeneity in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA of Labiostrongylus longispicularis, a parasitic strongylid nematode occuring in some species of kangaroo in different geographical regions of Australia. The results showed that most of the nematodes screened had different SSCP profiles, which were subsequently shown to correspond to polymorphisms and/or an indel in the ITS-2 sequence. These variable sites related mainly to unpaired regions of the predicted secondary structure of the precursor rRNA molecule. SSCP profiles could be used to distinguish L. longispicularis in Macropus robustus robustus (New South Wales) from L. longispicularis in Macropus robustus erubescens and Macropus rufus (South Australia). This difference corresponded to a transversional change in the ITS-2 sequence at alignment position 82. The study demonstrated clearly the effectiveness of SSCP analysis for future large-scale population genetic studies of L. longispicularis in order to test the hypothesis that L. longispicularis from different geographical regions represents multiple sibling species.

Animals↗
PubMed1999

Clonal identities and multiple isotype transcripts in hematological diseases revealed by a single-strand conformation polymorphism analysis of the immunoglobulin heavy chain messenger signals.

A single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified products of immunoglobulin (Ig) heavy chain rearrangements can be used to analyze B cell clonalities and clonal identities of B cells from different samples. However, the usefulness of the PCR-SSCP analysis is not fully assessed in B cell malignancies. For example, we did not know whether the PCR-SSCP method can be used to detect tumor-related clones in peripheral blood of patients with multiple myeloma and Waldenström's macroglobulinemia. In addition, because genomic DNA is used in the PCR-SSCP method, we could obtain no information about the isotype of the expanded B cell clone. In this study, we combined the reverse transcriptase (RT)-PCR of immunoglobulin heavy chain transcripts with an SSCP analysis and thus analyzed eight healthy individuals, five patients with B chronic lymphocytic leukemia, four patients with multiple myeloma and three patients with Waldenström's macroglobulinemia. Clonal B cell populations were detected as discrete bands in the RT-PCR-SSCP analysis that can be readily detected over the background of polyclonal rearrangements. Circulating tumor-related clones were detected in all but one peripheral blood sample from multiple myeloma and Waldenström's macroglobulinemia patients and B cell clones in peripheral blood and bone marrow from these patients showed a similar mobility on SSCP gel. Because the transcripts of different isotypes were separately analyzed, we could thus determine the isotype of B cell clones as well. When monoclonal Igs of different isotypes were detected in the individual samples, we analyzed the relationship of each monoclonal band by excising the band and then further analyzing it by a PCR-SSCP analysis. RT-PCR amplification in conjunction with the SSCP analysis is thus considered to be a useful method to detect and characterize the B cell clones in hematological diseases.

Aged↗
PubMed1978

Conformation-reactivity relationship for pyridoxal Schiff's bases. Rates of racemization and alpha-hydrogen exchange of the pyridoxal Schiff's bases of amino acids.

The role of stereoelectronic effects in controlling the reaction specificity of biological reactions involving pyridoxal phosphate-amino acid Schiff's bases was tested with nonenzymatic models. The rates of racemization and Halpha exchange of a series of pyridoxal-amino acid Schiff's bases were determined. The order of these rates does not parallel the predictions based solely on electronic or steric effect, but parallels the proportions of the reactive conformers (e.g., conformers with the Calpha--Halpha bond orthogonal to the pi system) estimated by CPK models. The special reactivity of the phenylalanine Schiff's base was consistent with a special conformation in which some type of pi--pi interaction increases the proportion of exchangeable conformers, thus further substantiating the role of conformation in governing the reactivity of the Calpha--Halpha bond. Furthermore, semiempirical calculations of the conformation about the Calpha00N bond were performed using the CAMSEA conformational analysis program. The results of conformational calculations are consistent with the results of conformational analysis by nuclear magnetic resonance. The order of reactivity of the the Calpha--Halpha bond of the SB dianion, pH 12.0, predicted by calculation based on stereoelectronic effects, though not quantatively parallel to the observed rate constants, is qualitatively in agreement with the experimental results.

Amino Acids↗
PubMed1989

Theoretical analysis of conformation and active sites of interferons.

Seven methods for prediction of protein secondary structures [1, 2, 14, 21, 22, 30, 31] were used for analysis of conformation of alpha- and beta-interferons (IFN-alpha and -beta). The results of the analysis indicate that the dominant secondary structure of IFNs-alpha and -beta is an alpha-helix. Five segments of the helix predicted by all the methods used have been identified. Using the method of Vonderviszt and Simon in IFNs-alpha and -beta two domains are predicted, the border between them lying at the region of amino acid (aa) residues 100-110. Comparison of primary structures of IFNs-alpha, -beta and -omega of different origin revealed two peculiar regions of conservative positions. In the first region, restricted to the N-terminal domain, 9 of 13 conservative positions are occupied by hydrophilic residues. In the second region, restricted to the C-terminal domain, all 8 conservative positions are occupied by hydrophobic residues. The C-terminal domain of the IFNs-alpha (aa residues 116-165) has been found to have a statistically valid homology (36%, probability of random coincidence is 5 x 10(-4) with prothymosin-alpha 1 (residues 1-47). The results of the theoretical analysis as compared to the experimental data available suggest the existence of at least two active sites in IFNs-alpha, beta and omega.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells.

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.

Adenosine Triphosphate↗
PubMedDate not supplied

Where are the GaPs? A rational approach to monomer acquisition and selection.

Gridding and partitioning (GaP) is a computational method for the classification and selection of monomers for combinatorial libraries. The molecules are described in terms of the pharmacophoric groups they contain and where those pharmacophoric groups can be located in three-dimensional space. The approach involves a detailed conformational analysis of each molecule. This conformational analysis is done within a common coordinate frame, thus enabling the monomers to be compared. The use of a partitioned space is central to this particular application as it facilitates the identification of regions of space which are not well represented by existing compounds. Several ways to extend the use of partitioned pharmacophore spaces are described. Applications of the approach in monomer acquisition and in library design are outlined.

Algorithms↗
PubMed2004

Severe factor XI deficiency in a Lebanese family: identification of a novel missense mutation (Trp501Cys) in the catalytic domain.

In this study, a Lebanese woman with severe factor XI deficiency as well as several unaffected family members were analysed. The F11 gene was screened by polymerase chain reaction amplification of all 15 exons, including intron-exon junctions followed by single-strand conformational analysis. Variant single-strand conformational analysis profiles were obtained for exon 13; sequencing of these products allowed the identification of a novel missense mutation (Trp501Cys) situated in the catalytic domain, in homozygosity in the proband.

Adult↗
PubMed2002

Pyrosequencing as an alternative to single-strand conformation polymorphism analysis for detection of N-ras mutations in human melanoma metastases.

BACKGROUND: Mutations in codons 12, 13, and 61 of the N-ras gene are common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras. METHODS: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively. RESULTS: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis and confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified. CONCLUSION: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.

Codon↗
PubMedDate not supplied

Frequent detection of human papilloma viruses in cervical dysplasia by PCR single-strand DNA-conformational polymorphism analysis.

BACKGROUND: To clarify the pathogenicity of multiple human papilloma virus (HPV) infection, we applied SSCP (single-strand DNA conformation polymorphism) analysis for cervical neoplastic lesions. MATERIALS AND METHODS: Two hundred and sixty-six cervical swab specimens from normal cervix (n=64), cervical dysplasia (n=95), carcinoma in situ (n=79) and cervical cancer (n=28), were studied by nested PCR-SSCP analysis using L1 consensus primers. RESULTS: In 95 samples of cervical dysplasia, HPV infection was detected in 98.9% (94 out of 95), multiple HPV infection was detected in 38.3% (36 out of 94). In 19 squamous cell carcinomas (SCC) and 9 adenocarcinomas, the detection rate of HPV infection was 84.2% (16 out of 19) and 55.6% (5 out of 9), respectively, and all HPV-positive cases showed infection of a single HPV, among which HPV 16 occupied 68.6% (11 out of 16) in SCC and HPV 18 occupied 100% (5 out of 5) in adenocarcinoma. CONCLUSION: Multiple HPV infections may be concerned with pathogenicity in cervical dysplasia; however, the single infection with only a few HPV types, such as type 16 in SCC and type 18 in adenocarcinoma, may play a role in cervical carcinogenesis.

Adenocarcinoma↗
PubMed1997

Single-strand conformation polymorphism analysis to detect p53 mutations: characterization and development of controls.

Single-strand conformation polymorphism (SSCP) analysis is widely used to prescreen mutations in p53 gene. However, standardization of SSCP to detect p53 mutations has rarely been pursued so far. We have developed complete conditions for a temperature-controlled nonradioactive SSCP for mutation detection in amplified p53 exons 4-8, where mutations frequently occur in human tumors. Easily obtainable and clearly distinguishable positive controls were developed by replacing the regular 5' primers in amplification with primers that include one to three mutated sites. Careful purification of the amplified products by gel electrophoresis appeared to be essential. The efficiency of the method was studied by using previously sequenced samples with p53 mutations and the various positive controls. The use of two temperatures (exon 4: 4 degrees C and 15 degrees C; other exons: 4 degrees C and 20 degrees C) in combination with other optimized conditions resulted in 98% efficiency in mutation detection, which was considered sufficient for routine screening.

Exons↗
PubMed1995

Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.

Bacteria↗
PubMed1993

Sequence variation in the noncoding region of human papillomavirus type 16 detected by single-strand conformation polymorphism analysis.

Human papillomavirus (HPV) type 16 variants were found by single-strand conformation polymorphism (SSCP) analysis of the noncoding region of the viral genome. Two sets of primers were used to analyze a 1018 bp region spanning nucleotides 7109-222. Twelve SSCP patterns were demonstrated among HPV 16 DNAs purified from 48 anal specimens from 24 homosexual men. Seven patterns were detected among 10 HPV 16 isolates from cervical carcinomas. In two pairs of sex partners, identical variants were recognized in each partner of the pair. Infection with two variants of HPV 16 from 2 specimens was observed in 1 of 21 subjects for whom there were multiple samples over time. DNA sequence analysis of 7 isolates confirmed that the SSCP technique showed polymorphisms only in fragments that had base substitutions and that all of these base substitutions resulted in detectable shifts in fragment mobility.

Anal Canal↗
PubMed1992

Construction and molecular dynamics simulation of calmodulin in the extended and in a bent conformation.

Analysis of sequence similarity and comparison of the three-dimensional (3D) structures of troponin C and calmodulin have revealed a sequence in the central helix of calmodulin with a high probability for bending. The three amino acids known to form a bend in the N-terminal portion of troponin C are also found in the central helix of calmodulin. The modelling of a bent calmodulin structure, using the dihedral angles of the three residues in the bend of troponin C as a 3D template, results in a conformation of calmodulin where the N- and C-terminal domains are able to form contacts. Dynamics simulations starting from the X-ray structure of calmodulin and from the modelled bent calmodulin were carried out to compare flexibility and correlated movements of Ca2+ in the binding loops. Both conformations of calmodulin remained stable during the period of simulation. In the simulation of calmodulin in the extended form, the motions of the Ca2+ atoms in the two domains (Ca2+1 and Ca2+2 in one domain, and Ca2+3 and Ca2+4 in the other) are correlated. In the simulation of the bent form, an additional correlation between the Ca atoms in the two different domains is observed. The results are compatible with the occurrence of a bent conformation of calmodulin in the presence of targets, and with increased Ca2+ affinity and cooperativity of the Ca(2+)-binding loops in the calmodulin-peptide complexes.

Calcium↗
PubMed2000

Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer.

Sensitivity of single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was reported to be lower in capillary zone electrophoresis (CZE) compared to conventional slab gel electrophoresis. We examined the effects of buffer ion type, pH, and temperature in an attempt to improve the mutation detectability in the SSCP-CZE mode. It was noted that, by utilizing short-chain polyacrylamide as sieving media while simultaneously lowering the temperature, there was no improvement of conformer detectability. On the contrary, there was a large increment in conformers' resolution by running samples in a lower-pH buffer system. The effects of different buffering ions and pH values were investigated. By using a new buffer system, consisting of 35 mM 2-(N-morpholino)propanesulfonic acid (MES), 30 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylene diaminetetraacetic acid (EDTA), pH 6.8, and keeping constant all the other conditions, such as temperature, sieving, applied voltage, capillary length, and inner diameter (ID), there was a remarkable improvement in resolution and the sensitivity became comparable to that of slab gel systems.

Buffers↗
PubMed1998

Selection of appropriate HIV-1 genomic regions for single-strand conformation polymorphism analysis of the diversity, modification, and transmission of HIV-1 quasispecies.

Single-strand conformation polymorphism (SSCP) analysis is a useful tool for studying viral quasispecies. Four regions within the HIV-1 genome were studied by means of SSCP analysis with the aim of determining which regions were the most informative for the study of HIV-1 transmission or for detection of changes in HIV-1 quasispecies populations. Nested polymerase chain reaction (PCR) was used to amplify V1, V2, V3 of the env gene, and the p2 region in the gag gene. In total, 114 plasma specimens from 79 individuals were tested, including serial specimens from 10 mother-infant pairs that were provided by the Women and Infants Transmission Study (WITS). HIV-1 in specimens that were PCR-positive with primer pair SK38/SK39 showed different percentages of positive signals with primer pairs for the four regions: V1, 63%; V2, 83%; V3, 88%, and p2, 100%. HIV-1 sequences in the p2 target region displayed the greatest degree of polymorphism. Analysis of serial specimens showed that the V1 target region was the most variable of the four regions studied and was the most appropriate region for monitoring changes in quasispecies populations. Of the four regions studied, p2 was the most informative for the study of HIV transmission, as shown by analysis of samples from documented cases of mother to infant HIV-1 transmission.

Female↗
PubMed2005

Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis.

AIMS: To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. METHODS AND RESULTS: By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. CONCLUSIONS: PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.

Bacteriological Techniques↗
PubMed1991

Detection of aberrations of the p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis.

Aberrations of the p53 gene and its transcript in ten human tumor-derived cell lines were investigated by single-strand conformation polymorphism analysis of polymerase chain reaction products. This method can detect loss of one of the two alleles and a point mutation in the remaining allele simultaneously. Aberrations were newly found in four tumor cell lines. Three cell lines had lost one of the alleles and had a point mutation in the other. These point mutations, resulting in amino acid substitutions, were in the region of the p53 gene, which is highly conserved in different species. All the mutated genes were expressed and produced mRNAs with point mutations. One cell line did not contain any detectable transcript of the p53 gene, although no structural aberration of the gene was detected in the regions analyzed. Most of the cell lines found to carry aberrations of the p53 gene in this work have previously been reported to have mutations in oncogenes or in a tumor suppressor gene other than the p53 gene. These results provide additional examples of multiple genetic changes in the genesis of human tumors.

Alleles↗
PubMed1992

Detection of DNA aberrations in human cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products.

We have developed a simple, sensitive method, single-strand conformation polymorphism (SSCP) analysis, to detect a single nucleotide substitution in a DNA fragment amplified and labeled by the polymerase chain reaction (PCR). Mobility shift of single-stranded DNAs due to their specific conformations on non-denaturing polyacrylamide gel electrophoresis can reveal DNA aberrations. By the PCR-SSCP analysis of DNAs from surgical specimens of human cancers, mutated ras genes (17%) and aberrations of tumor suppressor p53 gene (53%) including loss of one of the two alleles and a mutation in the remaining allele were detected in lung carcinomas and aberrations of both of the p53 and retinoblastoma (RB) genes were detected exclusively in advanced hepatocellular carcinomas.

Carcinoma, Hepatocellular↗