Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells

1987 ◽  
Vol 7 (3) ◽  
pp. 1267-1270
Author(s):  
J L Yang ◽  
V M Maher ◽  
J J McCormick
Keyword(s):  
Gel Electrophoresis ◽  
Shuttle Vector ◽  
Point Mutations ◽  
Linear Increase ◽  
Amber Mutation ◽  
Large Deletions ◽  
Base Substitutions ◽  
Bound Residues ◽  
Supf Gene ◽  
Two Populations

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.

scholarly journals Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells.

1987 ◽  
Vol 7 (3) ◽  
pp. 1267-1270 ◽  
Author(s):  
J L Yang ◽  
V M Maher ◽  
J J McCormick
Keyword(s):  
Gel Electrophoresis ◽  
Shuttle Vector ◽  
Point Mutations ◽  
Linear Increase ◽  
Amber Mutation ◽  
Large Deletions ◽  
Base Substitutions ◽  
Bound Residues ◽  
Supf Gene ◽  
Two Populations

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.

scholarly journals A novel general approach to eucaryotic mutagenesis functionally identifies conserved regions within the adenovirus 13S E1A polypeptide.

1986 ◽  
Vol 6 (5) ◽  
pp. 1487-1496 ◽  
Author(s):  
D Kimelman
Keyword(s):  
Shuttle Vector ◽  
Point Mutations ◽  
Mutant Gene ◽  
Adenovirus Type ◽  
Cellular Morphology ◽  
Morphological Alteration ◽  
Morphological Alterations ◽  
E1a Gene ◽  
Adenovirus Type 5

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.

scholarly journals Activation-induced Deaminase (AID)-directed Hypermutation in the Immunoglobulin Sμ Region

2002 ◽  
Vol 195 (4) ◽  
pp. 529-534 ◽  
Author(s):  
Hitoshi Nagaoka ◽  
Masamichi Muramatsu ◽  
Namiko Yamamura ◽  
Kazuo Kinoshita ◽  
Tasuku Honjo
Keyword(s):  
B Lymphocytes ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Variable Regions ◽  
Class Switch ◽  
Large Deletions ◽  
Activation Induced Deaminase

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch μ region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.

scholarly journals Mitochondrial DNA Somatic Mutations (Point Mutations and Large Deletions) and Mitochondrial DNA Variants in Human Thyroid Pathology

2002 ◽  
Vol 160 (5) ◽  
pp. 1857-1865 ◽  
Author(s):  
Valdemar Máximo ◽  
Paula Soares ◽  
Jorge Lima ◽  
José Cameselle-Teijeiro ◽  
Manuel Sobrinho-Simões
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Mutations ◽  
Point Mutations ◽  
Human Thyroid ◽  
Thyroid Pathology ◽  
Large Deletions ◽  
Dna Variants ◽  
Mitochondrial Dna Variants

Detecting Point Mutations by Denaturing-Gradient Gel Electrophoresis

2003 ◽  
pp. 705-716
Author(s):  
Stephen R. Dlouhy ◽  
Patricia Wheeler ◽  
James A. Trofatter ◽  
Peter J. Stambrook ◽  
Jay A. Tischfield
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Point Mutations ◽  
Gradient Gel Electrophoresis

A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells

Gene ◽  
1985 ◽  
Vol 38 (1-3) ◽  
pp. 233-237 ◽  
Author(s):  
Michael M. Seidman ◽  
Kathleen Dixon ◽  
Abdur Razzaque ◽  
Robert J. Zagursky ◽  
Michael L. Berman
Keyword(s):  
Mammalian Cells ◽  
Shuttle Vector ◽  
Point Mutations ◽  
Vector Plasmid

Comparison of Different Techniques for the Detection of FLT3-Mutations in Exons 14/15 and 20 in Patients with Acute Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2306-2306
Author(s):  
Christian Thiede ◽  
Jan Heidemann ◽  
Markus Andreas Schaich ◽  
Thomas Illmer ◽  
Gerhard Ehninger
Keyword(s):  
Tyrosine Kinase ◽  
Gel Electrophoresis ◽  
Point Mutations ◽  
Lower Sensitivity ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Molecular Abnormalities ◽  
Flt3 Mutations ◽  
Exon 20 ◽  
Acute Myeloid

Abstract Mutations of the FLT3 receptor tyrosine kinase are among the most important molecular abnormalities in patients with acute myeloid leukaemia (AML). Two types of mutations are predominant, internal tandem duplication mutations located in exons 14/15 and point mutations in exon 20, coding for the activation loop (codons 835/836) of the second tyrosine kinase domain (TKD). Numerous assays have been described for the detection of these abnormalities, including standard agarose gel electrophoresis, high resolution fragment analysis (Genescan), restriction fragment length polymorphism (RFLP) analysis and denaturing high performance liquid chromatography (DHPLC). Limited data exist on the capacity of these methods to detect FLT3 mutations. We compared agarose gel electrophoresis (AGE), Genescan analysis (GS) and DHPLC for the detection of FLT3 mutations in exon 14/15 and RFLP and DHPLC for the detection of mutations in exon 20 in a total of 1071 primary AML samples from patients treated in the AML96 protocol of the Deutsche Studieninitiative Leukämie (DSIL). The sensitivity of the ITD-detection was compared using dilutions of the FLT3-ITD pos. cell line MV4-11 in normal cells. Clinical samples which were found positive only in DHPLC were cloned and sequenced. Results: The dilution experiments indicated the highest sensitivity for the GS-method (0.1%) whereas the other techniques were less sensitive (AGE: 6.4%; DHPLC: 3.2%). Altogether 210/1071 (19.6%) cases scored positive in all three techniques for mutations in exon 14/15, 35 (3.3%) samples where positive only in the GS, 19 (1.8%) in GS and DHPLC and 14 (1.3%) in DHPLC only. Sequence analysis confirmed the presence of point mutations/small deletions within the juxtamembrane domain in 12/14 of those samples positive in DHPLC only. Mutational screening for the TKD domain revealed 64 positive cases with both techniques (6%), 14 (1.3%) cases were only positive in the DHPLC, 1 (0.1%) case was found by RFLP only. Interestingly, the majority of those samples positive with the DHPLC showed mutations outside the hot-spot region (codons 835/836), namely mutations in codons 837, 839, 841 and 842. Conclusions: Taken together these data indicate that more advanced techniques (GS; DHPLC) might be able to detect more mutations in the FLT3 gene than previously reported. Although AGE appears to detect most clinically relevant FLT3-ITD mutations, GS analysis might be more appropriate to screen for this lesion. DHPLC screening for ITD mutations has a lower sensitivity but is able to detect point mutations, the clinical relevance of which is currently unknown. In contrast DHPLC might be an alternative for the assessment of mutations within the TKD domain, in this cohort, about 20% more TKD mutations were found using this method. This higher sensitivity might be especially relevant in the context of novel targeted therapies. In addition, the data presented here are the first comprehensive report on the prevalence of unusual FLT3-mutations in a large cohort of patients.

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