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PubMed1980

Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis.

The site-directed bisulfite mutagenesis technique has been used to construct a specific mutation, am404, at nucleotide position 3124 in the simian virus 40 genome. The mutation was contained within a PstI restriction site (map position 0.27) and prevented cleavage by PstI at that position. Nucleotide sequence analysis of the mutagenized region indicated that only a single base pair change had occurred: a guanosine x cytosine leads to adenine x thymine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of simian virus 40 indicted that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. am404 could not replicate autonomously when transfected into monkey cells (BSC-40) but did replicate when it was cotransfected with the late deletion helper virus dl1007. On the basis of its position in the T-antigen, gene am404 should produce a T-antigen 24% shorter than the wild-type protein.

Antigens, Neoplasm↗
PubMed1999

Polymerase chain reaction-based deletion screening of bisulfite (sulfur dioxide)-enhanced gpt-mutants in CHO-AS52 cells.

In this study, we have examined the mutagenicity of bisulfite (sulfur dioxide) at the xathine-guanine phosphoribosyl transferase locus (gpt) in the pSV2 gpt-transformed CHO cell line, AS52. Our results provide evidence for bisulfite as a weak gene mutagen because the chemical at high doses and at high cytotoxicity causes a 4-fold increase in mutant frequency (MF) and less than a doubling of the gpt gene deletion frequency compared to control. We suggest that the increase of MF in bisulfite-treated cells results from bisulfite activity,as a comutagen, enhancing the induction effect of unknown endogenous or exogenous factors on spontaneous mutagenesis of AS52 cells. For the spontaneous, 5 mM bisulfite- and 10 mM bisulfite-enhanced spontaneous mutants in AS52 cells, the percentage of total deletion mutations of the gpt gene is 36%, 44% and 65%, respectively

Animals↗
PubMed1993

The discriminator base influences tRNA structure at the end of the acceptor stem and possibly its interaction with proteins.

For many tRNAs, the discriminator base preceding the CCA sequence at the 3' end is important for aminoacylation. We show that the discriminator base influences the stability of the 1.72 base pair onto which it is stacked. Mutations of the discriminator base from adenosine to cytidine or uridine make the cytidine residue in the C1-G72 base pair of mutant Escherichia coli initiator tRNAs more reactive toward sodium bisulfite, the single-strand-specific reagent. The activity of the enzyme Met-tRNA transformylase toward these and other mutant initiator tRNAs is also consistent with destabilization of the 1.72 base pair in vitro and in vivo. By influencing the strength of the 1.72 base pair, the discriminator base could affect the energetic cost of opening the base pair and modulate the structure of the tRNA near the site of aminoacylation. For some aminoacyl-tRNA synthetases and other proteins that interact with tRNA, these factors could be important for specific recognition and/or formation of the transition state during catalysis.

Acyltransferases↗
PubMedDate not supplied

Methylation analysis of the fragile X syndrome by PCR.

The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and downregulation of transcription. The molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene usually by Southern blot analysis. We describe a PCR-based method for the analysis of methylation of the FMR1 gene, which involves bisulfite treatment of DNA prior to amplification. Fifty-two normal and 48 affected, premutation, or mosaic males were analyzed in a blinded study by this method. A prospective study of 30 males suspected of fragile X was also performed. Amplification specific for the methylated FMR1 sequence was readily observed in all individuals with a full mutation, whereas all normal and premutation individuals showed only amplification-specific for the unmethylated sequence, thus, allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism was also detectable by this method. Methylation-specific PCR appears to be a rapid and reliable tool for the diagnosis of fragile X males.

Base Sequence↗
PubMed1974

A simplified method for study of RNA conformation--reaction of formylmethionine transfer RNA with [14C]methylamine-bisulfite.

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNA(fMet) in solution. The procedure involves conversion of cytosine residues to N(4)-[(14)C]methylcytosines by treatment with (14)CH(3)NH(2) and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces (14)C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA(fMet) has been found to contain only six reactive cytosines: C(1), C(16), C(17), C(35), C(75) and C(76). In addition, slow reaction at C(m) (33) was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA(fMet) obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.

Artifacts↗
PubMed1992

Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis.

Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.

Amino Acid Sequence↗
PubMed1984

Compatibility considerations in parenteral nutrient solutions.

Information on compatibility of nutrients and drugs with parenteral nutrient (PN) solutions is reviewed and evaluated. Precipitation of calcium phosphate when calcium and phosphate salts are added can be affected by pH, amino acid concentration, amino acid product, temperature, sequence of additives, specific salt used, and time since admixture; precipitate formation can occur gradually over 24 hours. Insulin is chemically stable in PN solutions, but adsorption to the infusion system can cause decreased availability. Poor delivery of vitamin A via PN solutions has been reported. The sodium bisulfite content of amino acid injections may cause degradation of thiamine, but studies simulating clinical use are needed. Folic acid stability in PN solutions has been demonstrated, and phytonadione appears to be stable. Drug administration via PN solutions may be advantageous when fluid intake is restricted or peripheral vein access is limited and in home PN therapy. Summarized are results of studies involving heparin, cimetidine hydrochloride, aminophylline, amphotericin B, iron dextran, hydrochloric acid, corticosteroids, narcotics, metoclopramide, digoxin, and fluorouracil. Many antibiotics are probably stable, especially when administered by co-infusion rather than by direct mixture in the PN solution container. When lipids are mixed in the same container with amino acid-dextrose solutions, compatibility and stability of electrolytes, vitamins, and trace elements must be reassessed. Practical research is needed, and availability of additives should be studied in specific patient populations and for specific PN formulations. Valid conclusions are dependent on careful study design.

Adrenal Cortex Hormones↗
PubMed2002

Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase.

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.

Acyltransferases↗
PubMed1982

Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.

Base Sequence↗
PubMed2006

Association of smoking, CpG island methylator phenotype, and V600E BRAF mutations in colon cancer.

BACKGROUND: Cigarette smoking has been associated with microsatellite instability in sporadic colon cancer. Most microsatellite-unstable colon cancers have widespread methylation of CpG islands (i.e., the CpG island methylator phenotype [CIMP]), and many of these tumors harbor the V600E BRAF mutation. We investigated whether the association between smoking and all colon cancers could be explained through induction of CIMP and/or BRAF mutations. METHODS: We evaluated 1315 case patients with colon cancer and 2392 control subjects in a population-based study. Demographic information, including smoking history, was obtained in an interview. Microsatellite instability was determined primarily by evaluation of the mononucleotide repeat BAT-26. CIMP was determined by sodium bisulfite modification of DNA followed by methylation-specific polymerase chain reaction amplification of CpG islands in hMLH1, p16, and MINTS1, -2, and -31. Tumors were scored as CIMP high (i.e., > or = 2 CpG islands methylated) or CIMP low (i.e., < 2 CpG islands methylated). BRAF V600E mutations were identified by sequencing. Logistic regression was used to quantify relationships among smoking, CIMP, and BRAF. All statistical tests were two-sided. RESULTS: Heavy smoking (i.e., > 20 cigarettes per day), compared with nonsmoking, was associated with an increased risk of CIMP-high colon cancer (odds ratio [OR] = 2.06, 95% confidence interval [CI] = 1.43 to 2.97) and also with BRAF V600E mutations (OR = 3.16, 95% CI = 1.80 to 5.54). The association between cigarette smoking and the risk of colon cancer was limited to the minority of tumors that were CIMP high and BRAF wild type or CIMP high and BRAF mutated (for heavy smokers, OR = 1.91, 95% CI = 1.23 to 2.97, and OR = 2.85, 95% CI = 1.53 to 5.29, respectively). All relationships above showed a statistically significant relationship to amount smoked (P(trend) < .001 for all, except that relationship with tumors that were CIMP high and BRAF wild type, for which P(trend) = .008) and were independent of microsatellite instability. CONCLUSIONS: Previously identified associations between smoking and colon cancer, whether microsatellite unstable or stable, appear to be explained by the association of smoking with CIMP and BRAF mutations.

Amino Acid Substitution↗
PubMed2005

Human neuroglobin protein in cerebrospinal fluid.

BACKGROUND: Neuroglobin is a hexacoordinated member of the globin family of proteins. It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole - time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA). Peptides were sequenced (PepSeq, MassLynx v3.5) and proteins identified using MASCOT (R). RESULTS: Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects. CONCLUSION: This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined.

Journal Article↗
PubMed2005

A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.

OBJECTIVE: To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. METHODS: Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. RESULTS: The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. CONCLUSION: Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

Cell Line, Tumor↗
PubMed1980

Structural changes in the glutamine-chargeable Escherichia coli transfer RNA-Trp produced by chemical modification with sodium bisulfite.

Glutamine-mischargeable tRNA produced by sodium bisulfite-treated Escherichia coli tRNA-Trp was isolated by dihydroxyboryl-cellulose affinity column chromatography. This tRNA was shown to have dual specificity tryptophan and glutamine, and, when charged with either amino acid, bound to ribosomes in response to the non-sense codon UAG but not in response to the tryptophan codon UGG. The results were consistent with the reported properties of Su+7 tRNA. The bisulfite-treated tRNA-Trp migrated as two bands during polyacrylamide gel electrophoresis. The faster moving band (band I) coincided with that of untreated tRNA-Trp. The slower moving band (band II) coincided with the glutamine-chargeable tRNA-Trp. Su+7 tRNA behaved like band II tRNA upon gel electrophoresis. Nucleotide sequence analysis showed that a cytidine-uridine transition occurred at the 1st or the 2n position of the anitcodon of band II tRNA. Band I and band II tRNAs differed from each other in their thermal melting profiles. It is suggested that the single base change in the anticodon is responsible for the altered conformation of band II tRNA.

Autoradiography↗
PubMed2000

Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media.

Sensitive, safe and easy-to-use probes for the detection of nucleic acids are urgently called for. To this end we are in the process of developing a fluorescence-based technique to work in homogeneous assay media. We have examined pyrene and fluorescein as fluorescent labels for natural DNA probes. A fraction of the cytosine residues of a single-stranded cDNA was randomly labelled with either pyrene or fluorescein using the bisulfite-catalyzed diamine reaction. Both fluorophores showed fluorescence quenching when the labelled probe was hybridized with its complementary strand and we describe the changes in steady-state fluorescence intensity that occurred upon hybridization. Our results demonstrate that pyrene quenching is more efficient than fluorescein quenching and thus pyrene-labelled probes are more sensitive for detecting and quantifying DNA from natural sources.

Animals↗
PubMed1992

Differential elimination of radioiodine from single- and double-stranded 125I-DNA.

[125I]Iododeoxyuridyl residues in heat-denatured I-DNA underwent deiodination in solutions of sodium bisulfite. In 0.5 M bisulfite containing 1 M trimethylamine, 92% of the iodine was released from denatured DNA in 2.5 min at 60 degrees C. By contrast iodine in native I-DNA remained stably bound under the same condition. The deiodination of [125I]I-deoxyuridine residues incorporated by enzymatic copying of oligomers 60 nucleotides in length was found to be pairing dependent. While 125I in iododeoxyuridine paired to deoxyadenosine in a complementary oligomer remained approximately equal to 90% covalently bound in the presence of 1 M bisulfite at 60 C, when the iodouridylate residue was mismatched greater than 96% was eliminated in 4 min. The presence of mismatches in the two nucleotides adjacent to a [125I]iododeoxyuridine/deoxyadenosine pair increased the elimination of iodine. The results indicate that the deiodination reaction might be applied for the rapid detection of variations in natural DNA sequences.

DNA↗
PubMed1984

Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity.

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.

Amino Acid Sequence↗
PubMed1992

Identification of a novel promoter in the replication control region of plasmid R6K.

A novel source of transcription has been detected in the replication region of plasmid R6K by using fusions involving the galK reporter gene. The -35 and -10 consensus RNA polymerase binding sites were identified in the region overlapping the binding sites for the R6K-encoded replication protein pi. Transcription from this promoter, designated P2, is repressed in vivo by pi-protein levels that are inhibitory for replication. Promoter-down mutations in P2 induced in vitro by bisulfite mutagenesis result in a reduced copy number of a beta-replicon but not of a gamma-replicon. Implications of the role of P2 in R6K replication are discussed.

Bacterial Proteins↗
PubMed1993

Heterogeneity of Porphyromonas gingivalis strains on fimbrillin gene locus by restriction fragment length polymorphism analysis.

The adherence of Porphyromonas gingivalis (P. gingivalis) to the periodontal tissue may be an initial step in the pathogenesis of periodontal disease, and fimbriae is believed to play an important role in such processes. However, the heterogeneity of the size, sequence and antigenic reactivity of the fimbriae have been reported. The aim of the present study was to investigate the heterogeneity of P. gingivalis strains on the fimbrillin gene locus by RFLPs analysis. Fourty-seven P. gingivalis strains including 5 reference strains were used in this study. The plasmid (pUC13Bg 12.1) with the insert of fimA381 was modified with bisulfite and used as the probe. The genomic DNAs from P. gingivalis were digested with the restriction endonuclease SacI and PstI, electrophoresed, transferred and hybridized with the DNA probe. All Sac I digests generated one major band and the band size was almost the same (ca 2.5-kb), except strain W50. The Pst I digests showed one or two major bands and could be divided into 9 groups based on the band patterns. Moreover, isolates from one patient showed different band patterns. By RFLPs analysis, genetic heterogeneity seems to exist within the fimbrillin gene locus of P. gingivalis strains. Such genetic heterogeneity may reflect the previously reported difference of P. gingivalis fimbriae and moreover a single patient could be infected with more than one genotype of P. gingivalis.

Adult↗