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PubMed1988

Conformational alterations detected by circular dichroism induced in the normal ras p21 protein by activating point mutations at position 12, 59, or 61.

Activation of the oncogenic potential of ras oncogenes occurs by point mutations at codons 12, 13, 59, 61, and 63 of the sequences that codify for its product, a 21-kDa protein designated as p21. This activation has been postulated by computer models as modifiers of the structure of the protein, which may alter its biochemical and biological activities. We have expressed in bacteria the normal ras p21 and five mutated p21 proteins with mutations at positions 12, 59, 61, 12 plus 59, and 12 plus 61. Purification was carried out by solubilization from bacterial pellets in 7 M urea and chromatography through a Sephadex G-100 column to obtain greater than 95% purified proteins. Circular dichroic (CD) spectra showed that the normal protein and that activated by substitution of Ala59 to Thr59 are very similar in their overall structure. By contrast, point mutations affecting either 12 or 61 residues substantially altered the structure of the proteins. When the parameters of Chen et al. [Biochemistry II, 4120-4131 (1972)] were applied to the CD spectra, both normal and thr59-mutated ras proteins showed a less organized structure than mutated proteins at position 12 or 61. Since the Thr59 mutant has more similar transforming activity than other activated proteins, but a GTPase activity similar to that of the normal protein, our results support the hypothesis that there is more than one mechanism of activation of the ras p21 protein. One of these mechanisms involves important structural alterations by point mutations at position 12 or 61 which reduce the GTPase activity of the protein. Another mechanism will be that induced by a substitution of Ala59 to Thr59 which does not substantially alter the protein conformation. A putative alternative mechanism for the activation of this mutant is discussed.

Cell Transformation, Neoplastic↗
PubMed1996

Technical note: use of a PCR-single strand conformation polymorphism (PCR-SSCP) for detection of a point mutation in the swine ryanodine receptor (RYR1) gene.

We used the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis for screening the point mutation (C1843 to T) in the porcine ryanodine receptor (RYR1) gene. The PCR products (659 bp) were heat-denatured and separated by polyacrylamide gel electrophoresis. On silver-stained gels, the point mutation within the RYR1 gene could be detected clearly by mobility shifts. The best conditions for detecting the point mutation were by using a 5% polyacrylamide gel without glycerol and loading at 3 degrees C. The RYR1 genotypes diagnosed by PCR-SSCP were identical to the genotypes diagnosed by restriction enzyme fragment length polymorphism in all cases examined (n = 606).

Animals↗
PubMed2001

"Low-risk" and "high-risk" HPV-infection and K-ras gene point mutations in human cervical cancer: a study of 31 cases.

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for "low-risk" (types 6 and 11) and "high-risk" (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. "Low-risk" HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of "high-risk" human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that "high-risk" HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.

Adenocarcinoma↗
PubMed2001

Point mutation detection technology for the new-century laboratory.

The READIT system represents the newest contribution to point mutation detection technology. Specifically, the system involves hybridizing DNA or RNA probes using phosphorylation chemistry and luciferase as the detection method. The two primary features that distinguish the READIT technology from the competition are its versatility and the ability of users to design its own probes. The automated genotype assignment and statistical analysis simplify the use of this system even more. In the year 2002 more than a million tests will be performed in molecular diagnostics with significant contributions from both research and private laboratories.(1) The most important attributes for laboratorians when reviewing a new technology are cost, accuracy, automation, and ease of use. The advent of unique point mutation detection technologies will certainly assist the role of the laboratory in demonstrating the positive impact of testing on future applications of these technologies. Cost savings, accuracy, simplicity, and flexible throughput are the must-have features of successful point mutation detection technologies for the new-century laboratory.

Journal Article↗
PubMed1996

Keratin 9 point mutation in the pedigree of epidermolytic hereditary palmoplantar keratoderma perturbs keratin intermediate filament network formation.

Keratins form an intracellular keratin filament network in keratinocytes. Point mutations in the epidermal keratins could lead to the disruption of keratin filament formation, developing skin diseases such as epidermolytic hereditary palmoplantar keratoderma (EHPPK). We found a G to A transition in keratin 9 (K9) cDNA, resulting in the substitution of glutamine for arginine at 162, in all patients of a pedigree of EHPPK. Transfection into MDCK cells and DJM-1 cells revealed that the plasmid CMX vector containing normal keratin 9 cDNA showed normal keratin network formation, whereas the vector with a G to A point mutated keratin 9 cDNA showed disrupted keratin filaments with droplet formation in the cells. These results indicate that the point mutation seen in our patients had a dominant-negative effect on keratin network formation.

Amino Acid Sequence↗
PubMed2006

Leber's hereditary optic neuroretinopathy (LHON) associated with mitochondrial DNA point mutation G11778A in two Croatian families.

Leber's hereditary optic neuroretinopathy (LHON) is manifested as a bilateral acute or subacute loss of central vision due to optic atrophy. It is linked to point mutations of mitochondrial DNA, which is inherited maternally. The most common mitochondrial DNA point mutations associated with LHON are G3460A, G11778A and T14484C. These mutations are linked with the defects of subunits of the complex I (NADH-dehydrogenase-ubiquinone reductase) in mitochondria. The G11778A mitochondrial DNA point mutation is manifested by a severe visual impairment. In this paper two Croatian families with the LHON G11778A mutation are presented. Three LHON patients from two families were younger males which had the visual acuity of 0.1 or below, the ophthalmoscopy revealed telangiectatic microangiopathy and papilloedema, while Goldmann kinetic perimetry showed a central scotoma. The mothers and female relatives were LHON mutants without symptoms, whereas their sons suffered from a severe visual impairment. Molecular diagnosis helps to explain the cause of LHON disease.

Adult↗
PubMed1994

Searching for the 1 in 2,400,000: a review of dystrophin gene point mutations.

The past few years have seen a rapid increase in our knowledge of naturally occurring mutations in the dystrophin gene. Although earlier studies were limited to gross rearrangement mutations, we are now in a position to draw lessons on the molecular etiology of the remaining one-third of cases of Duchenne and Becker muscular dystrophy (DMD, BMD) which are associated with small mutations. This paper reviews 70 published and unpublished small mutations in the dystrophin gene and asks what we can learn about their nature, their distribution, and approaches to their characterisation. Strikingly for such a well-conserved gene, missense mutations are extremely rare, and the vast majority of DMD point mutations, like the gross rearrangements, result in premature translational termination. It seems increasingly likely that almost all cases of DMD arise solely as a result of a reduction in the level of dystrophin transcripts, and we argue that > 95% of DMD mutations contribute nothing to the functional dissection of the dystrophin protein. Most of the few BMD point mutations presented here are missense mutations in the N-terminal or C-terminal domains or are splice-site mutations that probably act, like BMD deletions, via the production of in-frame, interstitially deleted transcripts.

DNA↗
PubMed1997

Point mutations within and outside the homeodomain identify sequences required for proboscipedia homeotic function in Drosophila.

The Drosophila homeotic gene proboscipedia (pb) encodes a homeodomain protein homologous to vertebrate HoxA2/B2 required for adult mouthparts formation. A transgenic Hsp70-pb (HSPB) element that rescues pb mutations also induces the dominant transformation of antennae to maxillary palps. To identify sequences essential to PB protein function, we screened for EMS-induced HSPB mutations leading to phenotypic reversion of the HSPB transformation. Ten revertants harbor identified point mutations in HSPB coding sequences. The point mutations that remove all detectable phenotypes in vivo reside either within the homeodomain or, more unexpectedly, in evolutionarily nonconserved regions outside the homeodomain. Two independent homeodomain mutations that change the highly conserved Arginine-5 in the N-terminal hinge show effects on adult eye development, suggesting a previously unsuspected role for Arg5 in functional specificity. Three additional revertant mutations outside the homeodomain reduce but do not abolish PB+ activity, identifying protein elements that contribute quantitatively to pb function. This in vivo analysis shows that apart from the conserved motifs of PB, other elements throughout the protein make important contributions to homeotic function.

Amino Acid Sequence↗
PubMed1993

c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons.

High-molecular-weight DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in one case. One of the normally localized c-Ha-ras allele termed RCC c-Ha-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed.

Alleles↗
PubMed1995

Role of point mutation of the K-ras gene in tumorigenesis of B6C3F1 mouse lung lesions induced by urethane.

In order to elucidate the role of point mutation of the K-ras gene in the tumorigenetic process of lung tumors, an experimental model of lung lesions in mice induced by the administration of urethane was used. A total of 135 B6C3F1 male mice, 6 weeks old, were given urethane in the drinking water at 0, 6, 60, 600 or 1200 ppm, and were then killed after varying periods of time. The lung lesions were histologically characterized as hyperplasia, adenoma and adenocarcinoma. Point mutations in codons 12 and 61 of the K-ras gene were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by using dideoxy sequencing analysis. K-ras gene mutation was identified in 9 (23.7%) of 38 lesions classified as hyperplasia, 31 (46.3%) of 67 adenomas, and 3 (50%) of 6 adenocarcinomas. The most frequent mutation was an AT-to-TA transversion at the second base of codon 61 and this pattern accounted for 65% of the three mutant forms observed. These results suggest that the point mutation of K-ras gene is involved in all stages of mouse lung tumorigenesis, i.e., activation of this gene can also influence the later stages of lung lesions.

Adenocarcinoma↗
PubMed1990

Identification of point mutations by mispairing PCR as exemplified in MERRF disease.

The point mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA) from patients with myoclonic epilepsy and ragged red fibers (MERRF) was quantitatively analyzed after digestion with the restriction endonuclease Nae I of the PCR amplified DNA. Since the point mutation is not part of a restriction site for a commonly available restriction endonuclease, the Nae I restriction site was introduced by PCR using a mispairing primer. The percentage of mutated mtDNA was determined in a few hairs of five members of an affected family by counting the radioactivity of the fragments after PCR amplification with labelled dATP.

Adolescent↗
PubMed1991

Activation of Ki-ras gene by point mutation in human liver angiosarcoma associated with vinyl chloride exposure.

Point mutations of c-ras genes were investigated in human angiosarcomas of the liver associated with occupational exposure to vinyl chloride. DNA prepared from either frozen or paraffin-embedded tissues was amplified by the polymerase chain reaction, and putative point mutations at codons 12, 13, and 61 of c-Ha-ras, c-Ki-ras, and N-ras were analyzed by dot-blot hybridization with allele-specific oligonucleotides. A G.C----A.T transition in the second nucleotide at codon 13 of the c-Ki-ras-2 gene was detected in 5 of 6 tumors. This mutation is likely a consequence of vinyl chloride-DNA adduct formation. It leads to the substitution of glycine by aspartic acid in the resulting p21 protein, a consistent amino acid substitution found so far in all types of human cancer exhibiting a codon 13-mutated Ki-ras gene.

Base Sequence↗
PubMedDate not supplied

An estimate on the effect of point mutation and natural selection on the rate of amino acid replacement in proteins.

We outline a method for estimating quantitatively the influence of point mutations and selection on the frequencies of codons and amino acids. We show how the mutation rate, i.e., the rate of amino acid replacement due to point mutation, can be affected by the codon usage as well as by the rates of the involved base exchanges. A comparison of the mutation rates calculated from reliable values of codon usage and base exchange probabilities with those that would be expected on the basis of chance reveals a notable suppression of replacements leading to tryptophan, glutamate, lysine, and methionine, and particularly of those leading to the termination codons. If selection constraints are neglected and only mutations are taken into account, the best agreement between expected and observed frequencies of both codons and amino acids is obtained for alpha = 1.13-1.15, where (Formula: see text). The "selection values" of codons and amino acids derived by our method show a pattern that partially deviates from others in the literature. For example, the selection pressure on methionine and cysteine turns out to be much more pronounced than expected if only the discrepancies between their observed and expected occurrences in proteins are considered. To estimate to what extent randomly occurring amino acid replacements are accepted by selection, we constructed an "acceptability matrix" from the well-established matrix of accepted point mutations. On the basis of this matrix "acceptability values" of the amino acids can be defined that correlate with their selection values. We also examine the significance of mutations and selection of amino acids with respect to their physicochemical properties and functions in proteins. The conservatism of amino acid replacements with respect to certain properties such as polarity can be brought about by the mutational process alone, whereas the conservatism with respect to other relevant properties--among them all measures of bulkiness--obviously is the result of additional selectional constraints on the evolution of protein structures.

Amino Acid Sequence↗
PubMed1998

Detection of p53 point mutations by single strand conformation polymorphism: analysis by capillary electrophoresis.

We have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrophoresis (CE-SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG-SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5' Primers were labeled with FAM (5-carboxyfluorescein) and 3' primers were labeled with JOE (2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein). CE-SSCP was performed using the Perkin Elmer ABI PRISM 310 Genetic Analyzer with GeneScan Software and the Beckman P/ACE 5510 CE equipped for laser-induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild-type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE-SSCP measurements were performed at temperatures ranging from 10 to 60 degrees C on a prototype instrument. For mutations measured at ambient temperature (25 degrees C), characteristic shifts in direction and magnitude were observed in the migration times of both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitude and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature-specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE-SSCP. In addition, by using CE, discrete intra-strand isoforms could be easily observed at different temperatures. The combination of mutation-specific temperature profiling and analysis of isoforms by CE-SSCP should be of help to the diagnostic community in the detection of genetic mutations.

DNA Mutational Analysis↗
PubMedDate not supplied

Point mutations of the c-H-ras gene in spontaneous liver tumors of transgenic mice carrying the human c-H-ras gene.

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-ras gene (rasH2). The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-ras codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversions, and a CAA to CGA transition at the second base of codon 61, respectively. No point mutations in the human c-H-ras transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-ras gene, but no point mutations were detected in codon 61 of the mouse c-H-ras gene. No mutations in human or mouse c-H-ras codon 61 were detected in altered cell foci or hepatocellular adenoma. These results indicate that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosacomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rasH2 Tg mice.

Animals↗
PubMed1995

Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.

Base Sequence↗
PubMed1987

A comprehensive collection of point mutations in the internal promoter of the adenoviral VAI gene.

Point mutations in the adenoviral VAI gene promoter were generated by in vitro mutagenesis with sodium bisulfite and were identified by three phenotypes: the disappearance of certain restriction endonuclease cleavage sites, altered rates of transcription in cell-free extracts, and altered mobility of the RNA product on denaturing acrylamide gels. 25 different point mutations were assayed for their effect on VAI transcription in HeLa S100 extracts. Mutations which change the transcriptional ability of the gene were localized in the internal control regions which are highly conserved among genes transcribed by RNA polymerase III. The magnitude of the change in transcription correlates with the degree of conservation in the consensus sequence at the site of the mutation. Most of the mutations either do not change the transcription rate or reduce transcription less than 6-fold. Two mutations severely reduce the amount of RNA produced, whereas three mutations increase the production of VAI RNA. A second site revertant that partially restores transcription to a mutant gene that produces no detectable VAI has also been isolated.

Adenoviridae↗
PubMed1995

The appearance of drug resistance-associated point mutations in HIV type 1 plasma RNA precedes their appearance in proviral DNA.

Point mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type-1 (HIV-1) have been associated with reduced sensitivity of the virus to inhibition by the antiretroviral drug zidovudine (ZDV). During therapy we have previously observed the accumulation of these mutations, at codons 41, 67, 70, 215, and 219 of the RT gene, in proviral DNA sequences from infected peripheral blood mononuclear cells (PBMCs) and in cell-free virus RNA. Using a quantitative point mutation assay (PMA) we have been able to quantify the proportions of mutant and wild-type sequences at these points in mixed populations of viral RNA and DNA derived from clinical samples. Thus we have confirmed that the mutations can be detected earlier in cell-free virus RNA than in PBMC proviral DNA and their accumulation in RNA precedes that in DNA. We have analyzed serial samples from 10 subjects undergoing zidovudine therapy and shown significantly higher levels of mutations in cell-free viral RNA than in PBMC proviral DNA at codons 41, 70, and 215 during the in vivo acquisition of the mutations. In the group of subjects studied the mean time delay between RNA and DNA reaching a given level of mutation was 25 days.

Antiviral Agents↗