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Results for “DNA Mutational Analysis”

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PubMed1991

Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli.

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.

Amino Acid Sequence↗

PubMed1992

A microtitre format point mutation assay: application to the detection of drug resistance in human immunodeficiency virus type-1 infected patients treated with zidovudine.

An assay for the analysis of point mutations in DNA fragments amplified by the polymerase chain reaction is described. The method was applied to the analysis of mutations leading to single amino acid changes in the reverse transcriptase gene of human immunodeficiency virus type-1 that are associated with decreased sensitivity of the virus to the effects of the nucleoside analogue zidovudine. The assay uses a microtitre format allowing large numbers of clinical samples to be analysed, and is a quantitative assay giving information on the relative proportions of wild-type and mutant sequence at the point being analysed. The analysis of mutations in codons 67, 70, 215, and 219 of the RT gene in samples taken at various time points after the commencement of zidovudine therapy shows a high variability in the rate at which these mutations arise and in the proportions of wild-type and mutant sequences observed. The method we describe has wide application to the analysis of other point mutations in clinical or research studies.

Base Sequence↗

PubMed1994

Comparative analysis of DNA mutations in lacI transgenic mice with age.

Transgenic mice have been developed recently that contain copies of the well-defined mutagenesis reporter gene, lacI, in an integrated bacteriophage-based shuttle vector. The lacI gene, which is present in all cells of the mouse, can be excised specifically from isolated genomic DNA and efficiently packaged into bacteriophage particles after the addition of packaging extracts. Mutations of the lacI gene are easily detected by the derepression of beta-galactosidase resulting in blue plaques in the presence of X-gal. Originally developed as a short-term in vivo mutagenesis assay to screen genotoxic agents, we have used this system to measure naturally occurring DNA mutations as a function of chronological age. There was a linear increase in the presence of phenotypic lacI mutants from birth to 24 months (r = 0.731, P < 0.001), such that 24-month-old mice have accumulated approximately fourfold more mutants than newborn pups. Molecular analysis of these spontaneously arising DNA mutations showed them to result predominantly from base substitutions (80.6-98.5%) that were equally distributed between transitions and transversions. However, lacI mutations in animals > 3 months of age demonstrated a higher percentage of mutations (12-19.4% vs. 1.2%) resulting from discriminable size changes (> 20 bp) than was observed for mice 1-2 months old. Sequence analysis of mutations resulting from a > 20 bp size change revealed them to be due to a duplication of adjacent lacI sequence. These results indicate that there is a gradual accumulation of DNA mutations with age and that the types of mutations also are influenced by the age of the animal.

Aging↗

PubMed1986

Mutational analysis of DNA sequences affecting the replication of defective polyomavirus variant D-50.

The genome of the defective polyomavirus variant D-50 consists of tandemly repeated DNA segments. The repeat unit corresponds to a 17% fragment of polyomavirus DNA (Griffin and Fried (1975) Nature 256, pp. 175-179). To allow mutational analysis, a monomer unit of D-50 DNA was cloned. After excision from the plasmid and ligation to form a random mixture of products, circular head to tail oligomers of the cloned segment were replicated in transfected cells. Those molecules had the same replication properties as original D-50 DNA. Deletion of base sequences assumed to be at the origin of DNA synthesis inhibited the replication completely, whereas a deletion of a segment at the junction of the tandem repeats had only a slight inhibitory effect. Mutation of potential coding sequences of the variant genome had a slight stimulatory effect on DNA synthesis, ruling out that D-50 expresses any protein that stimulates replication. In an attempt to construct variants similar to D-50, cells were transfected with polyomavirus DNA fragments including the sequences of the D-50 monomer unit. However, all these molecules replicated very slowly, suggesting the presence of an element that inhibited DNA synthesis. The combined data show that D-50 genomes can be reconstituted by ligation of monomer units and that the origin of DNA replication was the only essential element of the variant genome, whereas other elements had cis-acting auxiliary functions.

Base Sequence↗

PubMed2006

A new system for detecting mutations in arabidopsis thaliana and the mutational spectra resulting from ethylmethanesulfonate treatment.

A system was developed for the detection and analysis of mutations occurring on chromosomal DNA in plants. The plasmid pML4, carrying the Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into a shuttle vector, pCGN5138, to construct a plasmid which could be used for the transformation of plants. pML4 sequences were introduced into Arabidopsis thaliana mediated by Agrobacterium. The pML4 DNA was rescued from transgenic Arabidopsis plants exposed to mutagens, and the plasmids were introduced into Escherichia coli DH10B to isolate mutant clones. In this system, any form of inactivation mutation in the rpsL gene can be positively selected since it makes the E. coli cells resistant to streptomycin. Here we report that the system could detect the mutagenic effect of ethylmethanesulfonate (EMS). Further characterization of the mutants revealed that G:C to A:T transitions predominated among the EMS-induced mutations. This assay system is useful for the detection and analysis of mutations arising on chromosomal DNA in plants, and should be useful for evaluating analysis of the effects of environmental mutagens.

Arabidopsis↗

PubMed1994

Two NF1 mutations: frameshift in the GAP-related domain, and loss of two codons toward the 3' end of the gene.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders, and is due to mutations within the NF1 gene on chromosome 17q11.2. Only the middle 400 amino acids of the associated protein (neurofibromin) have a known function, comprising a GTPase-activating-protein (GAP) domain. The large gene size and the fact that approximately half of cases are due to new mutation render mutation analysis difficult. NF1 direct mutation characterization is important for development of DNA diagnostic procedures, analysis of phenotype/genotype correlations, and delineation of functions for specific domains of neurofibromin. We report two mutations detected using PCR amplification of individual exons followed by heteroduplex analysis. One is a single base deletion in exon 24 which is predicted to result in a protein truncated early in the GAP-related domain. The other is a 6-bp deletion in exon 39 which is predicted to result in loss of two amino acids in the mature protein near the carboxy-terminus. The exon 24 mutant allele was shown to be expressed by RNA PCR analysis. The exon 39 mutation suggests that those two amino acids are important in neurofibromin function, perhaps indicating a functional domain.

Adolescent↗

PubMed2003

RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund-Thomson patient.

Rothmund-Thomson syndrome (RTS) (OMIM 268400) is an autosomal recessive genodermatosis associated with genomic instability and increased risk of mesenchymal cancers. Mutations in the RECQL4 gene, encoding a protein of the family of Werner (WRN) and Bloom (BLM) helicases, have been identified in a subset of RTS patients. Apart from congenital poikiloderma, the clinical presentation of RTS is widely variable, raising the question of the possible existence of a second locus. Results herein reported on a sporadic Caucasian patient emphasize the concept that mutation analyses at both DNA and RNA level complement the genetic defect suggested by clinical and cytogenetic signs. The patient presented with typical congenital poikiloderma and bone defects and exhibited significant genomic instability in the peripheral blood karyotype. By RECQL4 DNA mutation analysis, he was found to carry a 1473delT (mut 5) on one allele and an AG to AC change at the 3'-splice site of exon 13 (a variant of mut 4) on the second allele. RT-PCR analysis of RECQL4 cDNA encompassing the entire helicase domain showed diffuse splicing defects indicating that the loss of a single 3'-splice signal motif disregulates the correct splice-site selection and affects the overall RNA processing. The presence of an unstable minisatellite which ends at 3'-splice site of IVS12 may enhance the mutation at this site. This genomic feature together with a number of short introns in the RECQL4 gene may account for the common missplicing of RECQL4 mRNA. While it is possible that defects of RECQL4 mRNA processing might account for part of the clinical variability observed for this syndrome, only a thorough analysis at both genomic and RNA level may allow a genotype-phenotype correlation in RTS patients, restricting the search of a second RTS locus to the specific patients.

Adenosine Triphosphatases↗

PubMed2001

INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas.

The INK4a-ARF (CDKN2A)- locus on chromosome 9p21 encodes for two tumour suppressor proteins, p16(INK4a) and p14(ARF), that act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in tumorigenesis of hepatocellular carcinoma (HCC), we analysed the alterations of p14(ARF), p16(INC4a) and p53. After microdissection, DNA of 71 hepatocellular carcinomas was analysed for INK4-ARF inactivation and p53 mutation by DNA sequence analysis, methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), mRNA expression and immunohistochemistry. In addition, microdeletion of p14(ARF) and p16(INC4a) were assessed by differential PCR. Inactivation of p14(ARF) was found in 11/71 cases (15%), alterations of p16(INK4a) occurred in 47/71 carcinomas (66%), which correlated with loss of mRNA transcription. Five tumours (7%) had homozygous deletions of the INK4a-ARF locus. We failed to detect specific mutations of both exons. P16(INK4a) methylation with an unmethylated p14(ARF) promotor appeared in 39 cases. Mutations of p53 were found in 30 of 71 HCC (42%), and only one of them harboured p14(ARF) inactivation. We failed to establish alterations of the INK4a-ARF locus or p53 status as independent prognostic factor in these tumours. Our data indicate, that p14(ARF) methylation occurs independently of p16(INK4a) alterations in a subset of HCC together with wild type p53. The INK4a-ARF-/p53-pathway was disrupted in 86% of HCC, either by p53 mutations or by INK4a-ARF inactivation, and may have co-operative effects in hepatocarcinogenesis.

Adult↗

PubMed1991

A tester system for detecting each of the six base-pair substitutions in Saccharomyces cerevisiae by selecting for an essential cysteine in iso-1-cytochrome c.

A collection of isogenic yeast strains that is specifically diagnostic for the six possible base-pair substitutions is described. Each strain contains a single, unique base-pair substitution at the Cys-22 codon of the CYC1 gene, which codes for iso-1-cytochrome c. These mutations encode replacements of the functionally critical Cys-22 and render each strain unable to grow on media containing nonfermentable carbon sources (Cyc-). Specific base-pair substitutions, which restore the Cys-22 codon, can be monitored simply by scoring for reversion to the Cyc+ phenotype. These strains revert spontaneously at very low frequencies and exhibit specific patterns of reversion in response to different mutagens. Only true (CYC1+) revertants were recovered after 7 days on selection medium. The following mutagen specificities were observed: ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, G.C----A.T; 4-nitroquinoline-1-oxide, G.C----T.A and G.C----A.T; diepoxybutane, A.T----T.A, A.T----G.C and G.C----T.A; 5-azacytidine, G.C----C.G. Methyl methanesulfonate induced all six mutations, albeit at relatively low frequencies, with preference for A.T----T.A and A.T----G.C. Ultraviolet light was the most inefficient mutagen used in this study, consistent with its preference for transition mutations at dipyrimidine sequences reported in other systems. This tester system is valuable as a simple and reliable assay for specific mutations without DNA sequence analysis.

Base Composition↗

PubMed1992

Use of suppressor analysis to identify DNA polymerase mutations in herpes simplex virus which affect deoxynucleoside triphosphate substrate specificity.

Herpes simplex virus DNA polymerase mutations which map in the N-terminal part of the protein and appear to alter deoxynucleoside triphosphate (dNTP) substrate specificity are described. These mutations suppress a drug hypersensitivity associated with the downstream mutation, Aphr10. We suggest that the mutant residues form part of the dNTP-binding site, a site previously thought to be confined to the C terminus.

Aphidicolin↗

PubMed2001

X-linked ocular albinism (Nettleship-Falls): a novel 29-bp deletion in exon 1. Carrier detection by ophthalmic examination and DNA analysis.

BACKGROUND: Mutations in the OA1 gene on the short arm of the X chromosome are known to cause X-linked ocular albinism (x1OA) in males. A four-generation family with this disorder, including asymptomatic carrier females, was investigated by molecular analysis of the OA1 gene. METHODS: DNA samples were available from 22 individuals of this family, including 6 affected males and 6 obligate carriers. The nine exons of the OA1 gene were amplified and further analyzed by SSCP and sequencing. RESULTS: A detailed clinical examination of the index patient and two female carriers showed the typical signs of ocular albinism. Visual evoked potential responses showed markedly asymmetrical responses from the two hemispheres in the affected person as well as in the carriers, as a result of misrouting and decussation of optic nerve fibers. Molecular genetic analysis demonstrated a previously undescribed 29-bp deletion at position 225-253 in exon 1 of the OA1 gene, which segregated in the family. CONCLUSION: Clinical examination combined with molecular genetic analysis enhances the potential for a precise diagnosis for persons at risk of x1OA and provide an accurate basis for genetic counseling.

Adult↗

PubMed1998

Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

Adult↗

PubMed1992

DNA sequence analysis of mutations induced by melphalan in the CHO aprt locus.

In previous work, we established that treatment with melphalan (L-phenylalanine mustard) produced a predominance of A.T-->T.A transversions in the Simian virus 40 (SV40)-based shuttle vector pZ189 during replication in human 293 cells. Mutations were induced with varying doses (4-12 microM) melphalan in the aprt gene of the hemizygous Chinese hamster ovary (CHO) cell line D422 to determine whether a similar mutation spectrum would be observed in an endogenous gene. DNA sequence alterations were determined for 39 spontaneous and 41 melphalan-induced independent mutant clones. Other than a predominance of transversions in both systems, the spectrum of melphalan-induced aprt mutations bears little resemblance to the spectrum observed in the supF gene of the shuttle plasmid pZ189. In aprt, mutations at G.C base pairs (bp) predominated (29 of 41 base substitutions). Significantly enhanced mutagenesis was observed at 5' G-G-C 3' and 5' G-G-C-C 3' sites in the aprt gene. Almost half of the melphalan-induced base substitutions occurred at 5' G-N-C 3' sequences, which are believed to be potential interstrand crosslink sites.

Adenine Phosphoribosyltransferase↗

PubMed1995

Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis.

DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.

Amino Acid Sequence↗

PubMed2004

Mutational analysis of the Drosophila DNA repair and recombination gene mei-9.

Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift.

Alleles↗

PubMed2003

Colon cancer-associated DNA mutations: marker selection for the detection of proximal colon cancer.

This study evaluates the potential ability of a specific panel of DNA mutations to identify right-sided colorectal carcinomas (CRCs) that would be missed by a flexible sigmoidoscopy (FS) screening program. This panel could then be applied to stool DNA analysis for noninvasive proximal CRC detection. A series of resected right-sided CRCs from 101 patients who had no left-sided advanced colonic neoplasms distal to the splenic flexure were analyzed. Tumor DNA was isolated from microdissected tumor sections. Deletions in the BAT-26 locus, a marker of microsatellite instability, and mutations at 19 loci spread among the p53, K-ras, and Apc genes were detected following PCR amplification. Mutations were identified in 83% of successfully amplified samples and were variably present in each of the target sites: p53 (42%), Apc (37%), K-ras (28%), and BAT-26 (24%). Mutations were identified across all Dukes stages (CIS/A 6/8 [75%], B 41/51[80%], C 30/32 (94%), and D 6/9 [67%]). Our data suggest that this 20-marker mutation panel may be associated with more than 80% of cancers undetectable by FS. The adjunctive use of stool DNA mutation analysis using this marker panel in FS CRC screening programs may significantly increase the detection of proximal CRC.

Adenocarcinoma↗

PubMed2000

Efficient recovery of DNA from peripheral blood for diagnostic analysis with a vacuum manifold.

BACKGROUND: The increasing clinical use of diagnostic DNA mutation analysis requires efficient isolation of DNA from peripheral blood. METHODS AND RESULTS: The use of a vacuum manifold to isolate DNA was evaluated and compared with a similar centrifugation-based DNA isolation technique. In PCR-based assays of five-point mutations, identical results were obtained with DNA isolated from peripheral blood using either centrifugation or a vacuum system. Minor modifications to PCR procedures were encountered. CONCLUSIONS: In the clinical setting, this vacuum-driven method of DNA isolation provides an efficient, useful alternative to conventional centrifugation-based DNA isolation from peripheral-blood specimens. Providing sufficient, stable DNA for multiple assays, it is easily implemented without highly specialized, expensive equipment and decreases the time spent isolating DNA from multiple samples. In addition, the potential for specimen contamination is reduced because there are fewer transfer steps.

Clinical Laboratory Techniques↗

PubMed1997

Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions.

We have developed an in vitro DNA polymerase forward mutation assay using damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. The quantitative method uses complementary strand hybridization to gapped duplex DNA molecules and chloramphenicol selection. This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We have examined the accuracy of DNA synthesis catalyzed by calf thymus polymerase alpha-primase, polymerase beta and exonuclease-deficient Klenow polymerase. Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repeat sequence within the HSV-tk locus. Treatment of the DNA template with N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar dose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-tk system is unique in that damage-induced mutagenesis can be analyzed both quantitatively and qualitatively in human cells, in bacterial cells and in in vitro DNA synthesis reactions at a single target sequence.

Alkylation↗