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Strength of the purifying selection against different categories of the point mutations in the coding regions of the human genome.

Using available Information on the total absolute size of the coding region of the human genome, data on codon usage and pseudogene-derived mutation rates for different single nucleotide substitutions we have estimated, for the human genome, the potential numbers of mutation events capable to produce: (1) nonsense; (2) missense (radical and conservative); (3) silent; (4) splice; and (5) protein-elongating (those changing wild-type stop codon into an amino acid encoding codon) mutations. We used the NCBI dbSNP database to retrieve data on the observed number of polymorphisms of each category. The fraction of polymorphisms in each category among all potential events in the genome depends on the strength of selection: the higher the rate of polymorphism, the weaker the selection. We used nonsense mutations as a referent group. Compared with nonsense mutations, we found that the relative selection coefficient against protein-elongating mutations was 21%, and the relative selection was 12% against missense mutations. Radical missense mutations were found to be four times more deleterious compared to conservative ones. Surprisingly, we found that silent mutations on average are not neutral; with the average harmfulness of 3% of nonsense mutations. Silent mutations may be deleterious when they affect splicing by creating cryptic donor-acceptor sites or by disturbing exonic splicing enhancers (ESESs). The average selection coefficient against splice mutations was 48% of that against nonsense mutations. Converting the relative selection coefficients into absolute ones using data on loss-of-function mutations in Saccharomyces cerevisiae and Caenorhabditis elegans, or by analysis of the expected frequency of mutations in the human genome, suggested that genetic drift could play a role in population dynamics of conservative missense and silent mutations.

Computational Biology↗

Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of Nudaurelia capensis omega virus capsid protein.

Protein subunits of several RNA viruses are known to undergo post-assembly, autocatalytic cleavage that is required for infectivity. Nudaurelia capensis omega virus (Nomega V) is one of the simplest viruses to undergo an autocatalytic cleavage, making it an excellent model to understand both assembly and the mechanism of autoproteolysis. Heterologous expression of the coat protein gene of Nomega V in a baculovirus system results in the spontaneous assembly of virus-like particles (VLPs) that remain uncleaved when purified at neutral pH. After acidification to pH 5.0, the VLPs autocatalytically cleave at residue 570, providing an in vitro control of the cleavage. The crystal structure of Nomega V displays three residues near the scissile bond that were candidates for participation in the reaction. These were changed by site-directed mutagenesis to conservative and nonconservative residues and the products analyzed. Even conservative changes at the three residues dramatically reduced cleavage when the subunits assembled properly. Unexpectedly, we discovered that these residues are not only critical to the kinetics of Nomega V autoproteolysis, but are also necessary for proper folding of subunits and, ultimately, assembly of Nomega V VLPs.

Animals↗

A novel point mutation in P450c17 (CYP17) causing combined 17alpha-hydroxylase/17,20-lyase deficiency.

CONTEXT: Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare cause of congenital adrenal hyperplasia and hypogonadism. Novel single amino acid changes in P450c17 provide potentially important insights into key structural domains for enzyme function. OBJECTIVE, DESIGN, AND SETTING: We report a novel missense mutation in P450c17 in a 17-yr-old female presenting with a malignant mixed germ cell tumor with yolk sac elements who demonstrated clinical and biochemical features of combined 17alpha-hydroxylase/17,20-lyase deficiency. METHODS: Quantitative urinary steroid analysis was performed by high resolution gas chromatography. All eight coding exons of CYP17 were PCR amplified and sequenced. The position of arginine at codon 96 was modeled using the CYP17 structure 2c17 (www.rcsb.org). The CYP17 genes were subcloned into pcDNA3, expressed in HEK-293 cells, and chromatographed. PATIENT AND RESULTS: 17alpha-Hydroxylase deficiency was confirmed by marked reductions in urinary and serum cortisol, androgens, and estradiol. Mutational analysis revealed a novel homozygous R96Q missense mutation in P450c17, affecting an amino acid in a key substrate-binding region of the enzyme, leading to complete inactivity. CONCLUSION: The description of a second missense mutation at codon 96 (R96W and R96Q) in the substrate-binding region of P450c17 provides strong evidence for the key role of this amino acid in 17alpha-hydroxylase/17,20-lyase function. An association between a malignant germ cell tumor and 17alpha-hydroxylase deficiency has not been reported previously, although the presence of gonadoblastoma in the ovary of a patient with this condition has recently been described.

Adolescent↗

Adaptive amplification and point mutation are independent mechanisms: evidence for various stress-inducible mutation mechanisms.

"Adaptive mutation" denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac(-)Escherichia coli cells on lactose medium induces Lac(+)revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500-10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac(+)cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v)lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.

Adaptation, Biological↗

A novel point mutation in the 3' flanking region of the DNA-binding domain of topoisomerase II alpha associated with acquired resistance to topoisomerase II active agents.

V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.

Amino Acid Sequence↗

Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation.

In 1994 we described a list of approximately 2500 point mutations in the p53 gene of human tumors and cell lines which we had compiled from the published literature and made available electronically through the file server at the EMBL Data Library. This database, updated twice a year, now contains records on 4496 published mutations (July 1995 release) and can be obtained from the EMBL Outstation-the European Bioinformatics Institute (EBI) through the network or on CD-ROM. This report describes the criteria for inclusion of data in this database, a description of the current format and a brief discussion of the current relevance of p53 mutation analysis to clinical and biological questions.

CD-ROM↗

Point-mutational MspI and Ile-Val polymorphisms closely linked in the CYP1A1 gene: lack of association with susceptibility to lung cancer in a Finnish study population.

In this study of 87 lung cancer patients, 23 patients with lung disease other than cancer, and 121 healthy controls, no association was found between the MspI restriction fragment length polymorphism (RFLP) of the CYP1A1 gene and lung cancer risk. In the lung cancer population, histological type, smoking, and occupational histories were also examined with respect to increased lung cancer risk. No association was found between the MspI RFLP in the CYP1A1 gene and any of these variables. This is in contrast to the results of an earlier report describing an association between the rare genotype m2m2 and susceptibility to lung cancer in a Japanese population; but another study in Norway found no such association. It is evident that, in the Nordic population, MspI polymorphism in the CYP1A1 gene does not indicate individual susceptibility to lung cancer. We also studied a new point mutation which has recently been closely linked to the MspI restriction site polymorphism in a Japanese study population. This mutation results in an isoleucine-valine amino acid replacement in the heme binding region of human CYP1A1. We obtained a similar linkage in our study, so the discrepancy between the Japanese and the Nordic MspI RFLP findings cannot be based on a different degree of linkage between these two point mutations.

Alanine↗

Detection of a point mutation using short oligonucleotide probes in allele-specific hybridization.

Two nonradioactive and simple procedures were developed to detect the A985G point mutation that causes medium-chain acyl-CoA deficiency. In both of these assays, short oligonucleotide probes were used in allele-specific hybridization combined with DNA amplification. The lower limit for a useful probe was found to be between 9 and 12 base pairs. Time-resolved fluorometry was utilized as the label technology and microtitration plates as the solid support. In one of the assay formats, probes labeled with europium and samarium chelates were used to simultaneously detect the mutant and normal alleles from the same hybridization reaction. In addition, the discrimination efficiency of different probes was characterized by cross-reactivity determinations and by measuring affinities of the probes towards fully complementary as well as towards mismatch-forming target oligonucleotides. All of the 80 coded patient samples analyzed were correctly typed in both of the assay formats used.

Alleles↗

[The point mutation of pre-C region HBV-DNA in sera from chronic hepatitis B].

To clarify the relationship between a point mutation of HBV-DNA Pre-C region and the serological feature of chronic hepatitis B, we determined the Pre-C region sequence of HBV-DNA obtained sera of 39 patients with chronic hepatitis B. The screening of the gene arrangement of Pre-C region of HBV-DNA was performed by a direct sequence method amplifying HBV-DNA by polymerase chain reaction methods. In 22 of HBeAg positive cases, a mutant type (the 28th codon changed from TGA to TAG: stop codon) was found in only one case. The other hand, In 17 of HBeAg negative cases, it was found in 6 cases with fluctuating ALT and DNA-polymerase levels. We concluded that mutant viral infections could be the main cause of HBeAg negative cases with fluctuating ALT and DNA-polymerase levels.

Adult↗

Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis.

This report describes a new genotyping method capable of detecting low-abundant point mutations in a homogeneous, separation-free format. The method is based on integration of oligonucleotide ligation with a semiconductor quantum dot (QD)-mediated two-color fluorescence coincidence detection scheme. Surface-functionalized QDs are used to capture fluorophore-labeled ligation products, forming QD-oligonucleotide nanoassemblies. The presence of such nanoassemblies and thereby the genotype of the sample is determined by detecting the simultaneous emissions of QDs and fluorophores that occurs whenever a single nanoassembly flows through the femtoliter measurement volume of a confocal fluorescence detection system. The ability of this method to detect single events enables analysis of target signals with a multiple-parameter (intensities and count rates of the digitized target signals) approach to enhance assay sensitivity and specificity. We demonstrate that this new method is capable of detecting zeptomoles of targets and achieve an allele discrimination selectivity factor >10(5).

Cell Line, Tumor↗

Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA.

Capillary gel electrophoresis (CGE) and polymer-based microelectrophoretic platforms were investigated to analyze low-abundant point mutations in certain gene fragments with high diagnostic value for colorectal cancers. The electrophoretic separations were carried out on single-stranded DNA (ssDNA) products generated from an allele-specific ligation assay (ligase detection reaction, LDR), which was used to screen for a single base mutation at codon 12 in the K-ras oncogene. The presence of the mutation generated a ssDNA fragment that was >40 base pairs (bp) in length, while the primers used for the ligation assay were <30 bp in length. Various separation matrices were investigated, with the success of the matrix assessed by its ability to resolve the ligation product from the large molar excess of unligated primers when the mutant allele was lower in copy number compared to the wild-type allele. Using CGE, LDR product models (44 and 51 bp) could be analyzed in a cross-linked polyacrylamide gel with a 1000-fold molar excess of LDR primers (25 bp) in approximately 45 min. However, when using linear polyacrylamide gels, these same fragments could not be detected due to significant electrokinetic biasing during injection. A poly(methylmethacrylate) (PMMA) microchip of 3.5 cm effective column length was used with a 4% linear polyacrylamide gel to analyze the products generated from an LDR. When the reaction contained a 100-fold molar excess of wild-type DNA compared to a G12.2D mutant allele, the 44 bp ligation product could be effectively resolved from unligated primers in under 120 s, nearly 17 times faster than the CGE format. In addition, sample cleanup was simplified using the microchip format by not requiring desalting of the LDR prior to loading.

Acrylic Resins↗

A point mutation in the cargo-binding domain of myosin V affects its interaction with multiple cargoes.

Class V myosins move diverse intracellular cargoes, which attach via interaction of cargo-specific proteins to the myosin V globular tail. The globular tail of the yeast myosin V, Myo2p, contains two structural and functional subdomains. Subdomain I binds to the vacuole-specific protein, Vac17p, while subdomain II likely binds to an as yet unidentified secretory vesicle-specific protein. All functions of Myo2p require the tight association of subdomains I and II, which suggests that binding of a cargo to one subdomain may inhibit cargo-binding to a second subdomain. Thus, two types of mutations are predicted to specifically affect a subset of Myo2p cargoes: first are mutations within a cargo-specific binding region; second are mutations that mimic the inhibited conformation of one of the subdomains. Here we analyze a point mutation in subdomain I, myo2-2(G1248D), which is likely to be this latter type of mutation. myo2-2 has no effect on secretory vesicle movement. The secretory vesicle binding site is in subdomain II. However, myo2-2 is impaired in several Myo2p-related functions. While subdomains I and II of myo2-2p tightly associate, there are measurable differences in the conformation of its globular tail. Based solely on the ability to restore vacuole inheritance, a set of intragenic suppressors of myo2-2 were identified. All suppressor mutations reside in subdomain I. Moreover, subdomain I and II interactions occurred in all suppressors, demonstrating the importance of subdomain I and II association for Myo2p function. Furthermore, 3 of the 10 suppressors globally restored all tested defects in myo2-2. This large proportion of global suppressors strongly suggests that myo2-2(G1248) causes a conformational change in subdomain I that simultaneously affects multiple cargoes.

Binding Sites↗

Fine mapping of mitochondrial RNAs derived from the mtDNA region containing a point mutation associated with MELAS.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disorder associated with heteroplasmic point mutations in the mitochondrial tRNA(Leu)(UUR) gene. While previous studies have shown that the MELAS mutation at nt-3243 results in impairments in mitochondrial protein synthesis and respiratory chain function, it was not clear whether these were associated with structural alterations in mature RNAs derived from transcription of the region containing the mutation. We have performed fine mapping and high-resolution. Northern analysis of RNAs from cybrids derived from two MELAS patients harboring the nt-3243 mutation. No differences in the size or steady-state levels of transcripts from the 16S rRNA, tRNA(Leu)(UUR), or ND 1 genes (which are contiguous in the mtDNA) were observed between cell lines containing mutated or wild-type mtDNAs. Therefore, it is not likely that the protein synthesis defects observed in cybrids with the MELAS-3243 mutation are directly caused by qualitative alterations in either transcription termination or processing of these mitochondrial RNAs.

Base Sequence↗

[New DNA diagnostic system for determination and identification of homo- and heterozygote point mutations].

A straightforward and effective PCR-based assay system is devised that allows one to reveal and identify homozygous and heterozygous point mutations. The system uses two sets of allele-specific primers. In one set, the 3'-nucleotide matches the allele under study so that the primer functions effectively only if the DNA contains the corresponding allele. To increase primer specificity, template-noncomplementary nucleotides are introduced near its 3'-end. The primers from another set invariably bear a 3'-terminal mismatch, and, in addition, the mutant nucleotides of the alleles under study form mismatches with the internal nucleotides of the primers. In such combination, the primer activity is suppressed if the DNA contains a homozygous mutation. The assay system devised was utilized to reveal the Leiden mutation in the gene for factor V of the human blood clotting system in patients with thrombophilia.

Alleles↗

Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain.

Malignant activation of the human trk proto-oncogene, a member of the tyrosine protein kinase receptor family, has been implicated in the development of certain human cancers, including colon and thyroid papillary carcinomas. trk oncogenes have also been identified in cultured cells transfected with various DNAs. In this study, we report the characterization of three in vitro-generated trk oncogenes, trk2, trk4, and trk5 (R. Oskam, F. Coulier, M. Ernst, D. Martin-Zanca, and M. Barbacid, Proc. Natl. Acad. Sci. USA 85:2964-2968, 1988), in an effort to understand the spectrum of mutational events that can activate the human trk gene. Nucleotide sequence analysis of cDNA clones of trk2 and trk4 revealed that these oncogenes were generated by a head-to-tail arrangement of two trk tyrosine protein kinase domains connected by a purine-rich region. These oncogenes code for cytoplasmic molecules of 67,000 (p67trk2) and 69,000 (p69trk4) daltons. In contrast, the product of the trk5 oncogene, gp95trk5, is a cell surface glycoprotein of 95,000 daltons. This oncogene was generated by a 153-base-pair in-frame deletion within sequences coding for the extracellular domain of the trk receptor. This activating deletion encompasses a triplet coding for one of the nine cysteine residues that the trk receptor shares with the product of the highly related trkB tyrosine protein kinase gene. Introduction of a single point mutation (TGT----AGT) in this codon resulted in a novel trk oncogene whose product, gp140S345, differs from the nontransforming trk proto-oncogene receptor in a single amino acid residue, Ser-345 instead of Cys-345. These results illustrate that multiple molecular mechanisms, including point mutation, internal deletion, and kinase domain duplication, can result in the malignant activation of the human trk proto-oncogene.

Amino Acid Sequence↗

Cytosine methylation associated with repeat-induced point mutation causes epigenetic gene silencing in Neurospora crassa.

Repeated DNA sequences are frequently mutated during the sexual cycle in Neurospora crassa by a process named repeat-induced point mutation (RIP). RIP is often associated with methylation of cytosine residues in and around the mutated sequences. Here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. A large proportion of strains that had undergone RIP of a linked duplication flanking a single-copy transgene, hph (hygromycin B phosphotransferase), showed partial silencing of hph. These strains were all heavily methylated throughout the single-copy hph sequences and the flanking sequences. Silencing was alleviated by preventing methylation, either by 5-azacytidine (5AC) treatment or by introduction of a mutation (eth-I) known to reduce intracellular levels of S-adenosylmethionine. Silenced strains exhibited spontaneous reactivation of hph at frequencies of 10(4) to 0.5. Reactivated strains, as well as cells that were treated with 5AC, gave rise to cultures that were hypomethylated and partially hygromycin resistant, indicating that some of the original methylation was propagated by a maintenance mechanism. Gene expression levels were found to be variable within a population of clonally related cells, and this variation was correlated with epigenetically propagated differences in methylation patterns.

Azacitidine↗

The erg-3 (sterol delta14,15-reductase) gene of Neurospora crassa: generation of null mutants by repeat-induced point mutation and complementation by proteins chimeric for human lamin B receptor sequences.

Null mutations were generated in the erg-3 gene of Neurospora crassa by repeat-induced point mutation (RIP). The mutants were viable, lacked ergosterol, were resistant to the steroidal glycoside alpha-tomatine and were sensitive to the phytoalexins pisatin and biochanin A. RIP was frequently associated with silencing of the hph gene located adjacent to the duplicated erg-3 sequence. The silencing of hph was reversible in the two cases examined and appeared to be due to the spread of cytosine methylation associated with RIP. The erg-3 mutant could be complemented by transformation with recombinant genes that encode proteins chimeric for amino acid sequences from the transmembrane (TM) domain of human lamin B receptor (LBR). This indicates that the LBR TM domain possesses delta14,15-reductase activity.

Amino Acid Sequence↗

Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum.

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.

Animals↗