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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↗
PubMed2005

An unusual family with Leber's hereditary optic neuropathy and facioscapulohumeral muscular dystrophy.

We performed an observational prospective analysis to study the clinical characteristics as well as a molecular genetic analysis of 17 members of a Thai family who had visual loss and/or muscle weakness. Their blood mitochondrial DNA were examined for the presence of the G11778A Leber's hereditary optic neuropathy (LHON) mutation. Facioscapulohumeral muscular dystrophy (FSHD) DNA analysis was performed in four members who had visual loss. Of 17 family members, the eight members who had the 11778 LHON mutation were all from branch 'a'. Three of these eight members had FSHD with a 17-27-kb deletion of a tandem repeat in the 4q35 subtelomere, and two had been clinically diagnosed as FSHD. Four of six examined members in branch 'b' showed muscular dystrophy clinically diagnosed as FSHD. No correlation of blood DNA analysis between LHON and FSHD in affected members was found. We describe the first family with FSHD and G11778A LHON in which a mutation in mitochondrial DNA at nucleotide position 11778 of branch 'a' was found to be the origin of the mutation.

Adolescent↗
PubMed2005

[The first molecular analysis of a Hungarian HNPCC family: a novel MSH2 germline mutation].

BACKGROUND: Hereditary nonpolyposis colorectal cancer is an inherited disease characterized by onset at an early age, an excess of synchronous and metachronous large bowel tumors and a variety of extracolorectal malignancies. Basal and squamous cell carcinomas of the skin are not customarily included in the tumor spectrum of the syndrome. The disease is caused by a germline mutation in one of the DNA mismatch repair genes, most commonly MSH2 or MLH1, and typically presents with microsatellite instability and frequent loss of mismatch repair protein expression in the tumor tissue. PATIENT: The case of a 62-year old woman who had a history of colon cancer at the age of 46 years, endometrial cancer at the age of 56 years, baso-squamous, and squamous cell cancer of the face at the ages of 53, 54, 62 and 58 years, respectively, and rectal cancer at 60 is reported. Her family fulfills the Amsterdam criteria for the diagnosis of hereditary nonpolyposis colorectal cancer. The baso-squamous cell, the squamous cell, the endometrial and the rectal cancers were assessed for the microsatellite instability status and the expression of the MSH2 and MLH1 mismatch repair proteins, and the p53 tumor suppressor protein by immunohistochemistry. Mutational screening using an automated capillary DNA sequencer was performed by the direct genomic sequencing of 17 fragments of the MSH2 gene, which covers promoter, all exons and flanking intronic regions. RESULTS: All cancers displayed microsatellite instability and were positive for the p53 protein. The immunohistochemical staining in the baso-squamous cell, the squamous cell, the rectal and endometrial cancers were negative for MSH2 and positive for MLH1 proteins. DNA sequencing analysis revealed a mutation c.2292G > A in exon 14 of the MSH2 gene, which is altering the 764. amino acid, the tryptophan to STOP codon (p.W764X). Thus the MSH2 protein is presumably truncated by 171 aminoacids. CONCLUSION: To the best of authors' knowledge, this is the first molecular characterization of a Hungarian hereditary nonpolyposis colorectal cancer family. According to the Human Mutation Database and International Collaborative Group of HNPCC Database, this mutation is novel, has not been reported previously. Cutaneous baso-squamous and squamous cell cancers may present as part of the HNPCC phenotype. Detection of the loss of mismatch repair protein expression and mismatch repair gene mutation mapping, represents a significant improvement of the diagnosis of this syndrome in Hungary. These examinations identify the mutation carriers who are at an increased risk of developing cancers.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Practical problems in detecting abnormal mitochondrial function and genomes.

Mitochondrial respiratory chain dysfunction causes a wide range of primary diseases in adults and children, with highly variable organ involvement. Diagnosis involves weighing evidence from a number of sources, including the clinical presentation, metabolic measurements in vivo, imaging studies, analysis of respiratory chain function or enzyme activities in vitro, studies of mitochondrial morphology after biopsy, and mitochondrial (mt) DNA mutation analysis. Irrespective of the category of the information, it can be difficult to determine whether abnormal results are due to primary defects of the respiratory chain or to practical problems that complicate the diagnostic methodology. This review describes six sources of such problems: genetic complexity, tissue and temporal variation, methodological limitations, secondary effects, logistical issues, and questions of interpretation. When these issues are all addressed, a reliable categorization of the diagnosis as definite, probable, or possible respiratory chain defect becomes possible.

DNA Mutational Analysis↗
PubMed1999

Applications of short-chain polydimethylacrylamide as sieving medium for the electrophoretic separation of DNA fragments and mutation analysis in uncoated capillaries.

In capillary electrophoresis (CE), separation of DNA fragments is usually performed in covalently coated capillaries. Recent studies have demonstrated that certain polymers form a dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA-capillary wall interactions. We developed a simple method for the synthesis of short-chain polydimethylacrylamide (PDMA) using isopropanol as a chain transfer agent. Capillary (<75 microm internal diameter) filling and replacement of this low-viscosity (14 cP at 4% PDMA) self-coating medium were easily carried out by commercial CE instruments. Using PDMA and uncoated capillaries, we first examined the separation of phi X174 HaeIII DNA digests and observed that the stability of the dynamic coating was markedly better at pH 7.8 than at pH 8.3. At this lower pH and nondenaturing conditions, high resolution of the phi X174 HaeIII DNA digests was obtained for more than 850 injections in the same capillary. We then exploited this sieving medium in CE using multiple approaches for mutation analysis of clinical DNA samples including separation of restriction enzyme cleavage products, analysis of single strand conformation polymorphisms, and simultaneous detection of several mutations using multiplex allele-specific PCR amplification. Our results demonstrate that CE in uncoated capillaries using PDMA as sieving medium is a simple, versatile, and reliable strategy for separation and mutation analysis of clinical DNA samples.

Acrylic Resins↗
PubMed1997

Identification and characterization of the human homologue of SH3BP2, an SH3 binding domain protein within a common region of deletion at 4p16.3 involved in bladder cancer.

In a search for candidate tumor suppressor genes within a 30-kb common region of deletion previously identified in bladder cancer cell lines, we isolated a 2.4-kb cDNA clone comprising 13 exons that spanned approximately 16 kb of genomic DNA. Mutation analysis was carried out by single-strand conformation polymorphism analysis on DNA from 12 bladder carcinoma cell lines and 26 bladder tumors with LOH on chromosome 4p. Direct sequencing of the transcript in 4 bladder carcinoma cell lines with deletions in this region was also carried out. Two polymorphisms in exons 2 and 5 were identified, but no tumor-specific mutations were found. Sequence analysis identified a high degree of homology with the mouse sh3bp2 gene, which is abl-binding, suggesting that this gene is the human homologue. The predicted amino acid sequence of the putative gene product contains a Src homology 2 domain, a Src homology 3 binding domain, and a pleckstrin homology domain, suggesting a possible role in signal transduction. No evidence was found to indicate that SH3BP2 is the tumor suppressor gene at 4p16.3 involved in bladder cancer. However, this study has identified an interesting human gene that is a potential negative regulator of the abl oncogene.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Analysis of mutations at the fragile X locus using the DNA probe Ox1.9.

In this study, 40 families segregating for fragile X [fra (X)] syndrome were examined for the presence of a mutation within the FMR-1 gene. Using the DNA probe Ox1.9, both carriers and affected individuals were found to contain an insertion/amplification-type of mutation with somatic instability. Variability in the size of the mutation, which ranged from less than 0.2 kb to approximately 13 kb, was observed both between individuals (even from the same family) and within individuals, who showed a smear rather than a discrete band(s) on Southern blot analysis. Transmission of the mutation by males resulted in little change of its size, while transmission by females usually resulted in an increase in size. Correlations were observed between the size of inserted/amplified DNA and the level of chromosome fragility and the presence or absence of mental impairment. Overall, a mutation was detected in 66 of 67 (99%) clinically affected males, in 12 of 13 (92%) transmitting males and in 95 of 112 (85%) carrier females. Equivocal results were obtained in 12 (11%) of the carrier females. No mutation was detected in 58 females and 33 males predicted to be normal by linkage, or in one female and 36 normal control males. These results strongly suggest that the mutation detected by Ox1.9 is closely associated with the cytogenetic and clinical expression of fra (X) syndrome. Additionally, the use of this probe along with other probe/enzyme combinations should provide a sensitive clinical assay for the detection of carriers of fra (X) syndrome.

Cytogenetics↗
PubMed2001

Gene expression microarrays: a 21st century tool for directed vaccine design.

DNA microarray technology is a new and powerful tool that allows the simultaneous analysis of a large number of nucleic acid hybridization experiments in a rapid and efficient fashion. The development of the DNA microarray chip has been driven by modern techniques of microelectronic fabrication, miniaturization and integration to produce what is referred to as "laboratory-on-chip" devices. The application of DNA chip technology includes the comprehensive analysis of multiple gene mutations and expressed sequences with regard to newer drug designs, host-pathogen interactions and the design of new vaccines. An advantage of microarray technology is that it can assist researchers to better define and understand the expression profile of a given genotype associated with disease, adverse effects from exposure to certain stimuli, or the ability to understand or predict immune responses to specific antigens. This paper briefly reviews DNA microarray technology and its implications with special reference to vaccine design. The technical aspects comprising array manufacturing and design, array hybridization, formatting, scanning and data handling are also briefly discussed.

Animals↗
PubMed1989

A system for the analysis of yeast ribosomal DNA mutations.

To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.

Base Sequence↗
PubMed2000

Diagnosis of quantitative mitochondrial DNA defects by rapidly prepared whole mitochondrial DNA probe.

Analysis of disease-causing mutations in mitochondria genome requires rapid and reliable genetic approaches. However, the preparation of mitochondrial DNA (mtDNA) probe used for the determination of quantitative and qualitative mtDNA defects is time-consuming, cumbersome, and requires complicated instrumentation. To overcome the difficulties encountered during isolation and purification of mtDNA, the authors developed an alternative method based on polymerase chain reaction (PCR) amplification of whole mtDNA genome. In this study, they show that PCR-amplified and fluorescein-labeled mtDNA probe makes it possible, through Southern blot analysis, to identify quantitative defect of mtDNA. The results indicate that mtDNA probe can be prepared rapidly by PCR amplification and used to determine the level of mtDNA in the patients with mitochondrial diseases.

Blotting, Southern↗
PubMed1994

DNA sequence analysis of spontaneous and gamma-radiation (anoxic)-induced lacId mutations in Escherichia coli umuC122::Tn5: differential requirement for umuC at G.C vs. A.T sites and for the production of transversions vs. transitions.

Escherichia coli umuC122::Tn5 cells were gamma-irradiated (137Cs, 750 Gy, under N2), and lac-constitutive mutants were produced at 36% of the wild-type level (the umuC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacId mutations was very similar to that for the wild-type strain). The specific nature of the umuC strain's partial radiation mutability was determined by sequencing 325 radiation-induced lacId mutations. The yields of radiation-induced mutation classes in the umuC strain (as a percentage of the wild-type yield) were: 80% for A.T-->G.C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A.T-->C.G transversions, 36% for G.C-->A.T transitions, 25% for multi-base deletions, 21% for A.T-->T.A transversions, 11% for G.C-->C.G transversions, 9% for G.C-->T.A transversions, and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umuC strain is near-normal for A.T-->G.C. transitions, single-base deletions and possibly A.T-->C.G transversions; is generally deficient for mutagenesis at G.C sites and for transversions, and is grossly deficient in multiple mutations. Damage at G.C sites seems more difficult for translesion DNA synthesis to bypass than damage at A.T sites, and especially when trying to produce a transversion. The yield of G.C-->A.T transitions in the umuC strain (36% of the wild-type level) argues that abasic sites are involved in no more than 64% of gamma-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD' proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G.C vs. A.T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.

Bacterial Proteins↗
PubMed2003

A common mutation and a novel mutation in Japanese patients with van der Knaap disease.

Van der Knaap disease, or megalencephalic leukoencephalopathy with subcortical cysts (MLC), is an autosomal recessive disorder clinically characterized by macrocephaly, ataxia, spasticity, and mental decline. Magnetic resonance imaging (MRI) shows swollen brain with diffuse white-matter abnormalities and subcortical cysts, particularly in the anterior-temporal region. Recently, the MLC1 gene was identified as the gene responsible for this disorder, and mutations in this gene were described in several patients. We studied three Japanese patients with van der Knaap disease at the molecular genetic level. Two of them were homozygous for a previously-described mutation, S93L, and one was a compound heterozygote for S93L and a novel mutation, 452-468del+g, which leads to frameshift with a premature termination codon. Combining our data with previous reports allowed us to estimate the molecular genetic basis of this disorder in seven Japanese patients. In summary, S93L was observed in six of seven (85.7%) patients at least in one allele, and ten of 14 (71.4%) alleles had this mutation. Therefore, S93L appears to be fairly frequent in Japanese patients with van der Knaap disease, and analysis for this mutation in DNA isolated from leukocytes would provide for an easy and precise diagnosis of this disorder in Japanese patients.

Adult↗
PubMed2001

The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas.

A comprehensive analysis of somatic and germline mutations related to DNA mismatch-repair (MMR) genes can clarify the prevalence and mechanism of inactivation in colorectal carcinoma (CRC). In the present study, 257 unselected patients referred for CRC resection were examined for evidence of defective DNA MMR. In particular, we sought to determine the frequency of hereditary defects in DNA MMR in this cohort of patients. MMR status was assessed by testing of tumors for the presence or absence of hMLH1, hMSH2, and hMSH6 protein expression and for microsatellite instability (MSI). Of the 257 patients, 51 (20%) had evidence of defective MMR, demonstrating high levels of MSI (MSI-H) and an absence of either hMLH1 (n=48) or hMSH2 (n=3). All three patients lacking hMSH2, as well as one patient lacking hMLH1, also demonstrated an absence of hMSH6. DNA sequence analysis of the 51 patients with defective MMR revealed seven germline mutations-four in hMLH1 (two truncating and two missense) and three in hMSH2 (all truncating). A detailed family history was available for 225 of the 257 patients. Of the seven patients with germline mutations, only three had family histories consistent with hereditary nonpolyposis colorectal cancer. Of the remaining patients who had tumors with defective MMR, eight had somatic mutations in hMLH1. In addition, hypermethylation of the hMLH1 gene promoter was present in 37 (88%) of the 42 hMLH1-negative cases available for study and in all MSI-H tumors that showed loss of hMLH1 expression but no detectable hMLH1 mutations. Our results suggest that, although defective DNA MMR occurs in approximately 20% of unselected patients presenting for CRC resection, hereditary CRC due to mutations in the MMR pathway account for only a small proportion of patients. Of the 257 patients, only 5 (1.9%) appear to have unequivocal evidence of hereditary defects in MMR. The epigenetic (nonhereditary) mechanism of hMLH1 promoter hypermethylation appears to be responsible for the majority of the remaining patients whose tumors are characterized by defective DNA MMR.

Adaptor Proteins, Signal Transducing↗