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PubMed1980

Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis.

The site-directed bisulfite mutagenesis technique has been used to construct a specific mutation, am404, at nucleotide position 3124 in the simian virus 40 genome. The mutation was contained within a PstI restriction site (map position 0.27) and prevented cleavage by PstI at that position. Nucleotide sequence analysis of the mutagenized region indicated that only a single base pair change had occurred: a guanosine x cytosine leads to adenine x thymine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of simian virus 40 indicted that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. am404 could not replicate autonomously when transfected into monkey cells (BSC-40) but did replicate when it was cotransfected with the late deletion helper virus dl1007. On the basis of its position in the T-antigen, gene am404 should produce a T-antigen 24% shorter than the wild-type protein.

Antigens, Neoplasm↗
PubMed1998

Functional analysis of the CAAT box in the major late promoter of the subgroup C human adenoviruses.

Comparisons among sequences predicted to encode the major late promoter (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation suggests that the CAAT box plays an important role in the function of the MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, in terms both of quantitative levels of transcription and of specificity, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically. The CAAT5 mutation had no discernible phenotype by itself but when coupled with the previously described USF0 mutation, which disrupts binding of the upstream stimulating factor (USF) but is otherwise phenotypically silent, gave rise to virus with a severe replication deficiency. Nuclear run-on assays showed that transcription initiation at the mutant MLP was significantly reduced compared with that of the wild type or the virus containing CAAT5 alone. Replication of the double mutant was lower than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further lowered the binding of transcription factor CP1 (also called CBF, NF-Y). Replacement of the CAAT box by an ATF binding site or an OCT1 binding site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial restoration of the wild-type phenotype. The failure to restore the functional redundancy normally exhibited by the CAAT box and the proximal upstream activating element is consistent with the idea that in the adenovirus MLP the CAAT box is preferred over others as the distal transcriptional element.

Adenoviruses, Human↗
PubMed2002

Fast phylogenetic methods for the analysis of genome rearrangement data: an empirical study.

Evolution operates on whole genomes through mutations that change the order and strandedness of genes within the genomes. Thus analyses of gene-order data present new opportunities for discoveries about deep evolutionary events, provided that sufficiently accurate methods can be developed to reconstruct evolutionary trees. In this paper we present two new methods of character coding for parsimony-based analysis of genomic rearrangements: one called MPBE-2, and a new parsimony-based method which we call MPME (based on an encoding of Bryant), both variants of the MPBE method. We then conduct computer simulations to compare this class of methods to distance-based methods (NJ under various distance measures). Our empirical results show that two of our new methods return highly accurate estimates of the true tree, outperforming the other methods significantly, especially when close to saturation.

Animals↗
PubMed1998

Oxidized, deaminated cytosines are a source of C --> T transitions in vivo.

The most common base substitution arising from oxidative damage of DNA is a GC --> AT transition. In an effort to determine the oxidized lesion(s) that gives rise to this mutation, the mutagenicity of three oxidized cytosines, 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, were investigated in Escherichia coli. An M13 viral genome was constructed to contain a single oxidized cytosine at a specific site. Replication in vivo of the single-stranded genomes yielded mutation frequencies of 0.05%, 83%, and 80% for 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, respectively. The predominant mutation observed was C --> T. A model for C --> T oxidative mutagenesis is suggested in which initial cytosine oxidation is followed by deamination to a poorly repaired uracil derivative that is strongly miscoding during replication.

Cytosine↗
PubMed2002

Evidence for the involvement of a hematopoietic progenitor cell in systemic mastocytosis from single-cell analysis of mutations in the c-kit gene.

Mast cells are derived from multipotential hematopoietic progenitors and are clonally increased in systemic mastocytosis, a disease associated with point mutations of codon 816 (most commonly Asp816Val) of c-kit. To study the lineage relationship and the extent of expansion of cells derived from the mutated clone, we examined the occurrence of the Asp816Val c-kit mutation in genomic DNA of individual sorted peripheral blood T cells, B cells, and monocytes in patients with indolent systemic mastocytosis. The mutation was detected in varying frequencies in the genomic DNA of individual B cells and monocytes and bone marrow mast cells in patients with extensive disease. In B cells, the immunoglobulin repertoire was polyclonal, indicating that the mutation occurred before V(H)/(D)/J(H) recombination. These results show that mastocytosis is a disorder of a pluripotential hematopoietic progenitor cell that gives rise to B cells and monocytes in addition to mast cells and that the affected clone shows variable expansion in these lineages in the peripheral blood of patients with systemic mastocytosis.

Adult↗
PubMed1967

Genetic mapping of mutations affecting phosphoglucose isomerase and fructose diphosphatase in Escherichia coli.

The loci on the Escherichia coli genome of mutations affecting phosphoglucose isomerase (pgi) and fructose diphosphatase (fdp) have been determined by conjugation and transduction experiments, chiefly three-factor crosses. The loci for these two constitutive enzymes of central intermediary metabolism lie in the same region of the chromosome, but they are not cotransducible. The order of some markers in this region of the chromosome is arg(ACFH) metA pgi malB uvrA fdp pyrB thr.

Chromosome Mapping↗
PubMed2001

Mutational patterns in the HIV genome and cross-resistance following nucleoside and nucleotide analogue drug exposure.

A variety of key mutations in HIV reverse transcriptase (RT) have been associated with nucleoside reverse transcriptase inhibitor (NRTI) exposure, which give rise to a diverse range of effects in terms of altered drug susceptibilities, viral replicative capacity and RT biochemistry. There are three basic mechanisms of resistance conferred by specific mutations in the coding region of RT. The first is drug discrimination, whereby a particular drug or drugs are either selectively excluded from uptake or from the RT-primer-template catalytic complex. Drug discrimination is, for the most part, relatively specific for individual drugs. Repositioning of the template-primer to prevent a catalytically competent complex in the presence of a bound drug molecule has also been observed in some instances, and forms a second mechanism. The third, and potentially most significant for long-term efficacy of the NRTIs, is pyrophosphorolysis, the primary mode of resistance to zidovudine. Mutations selected by this drug or stavudine serve to elevate the natural rate of the reverse reaction for RT. Pyrophosphorolysis uncouples the last nucleoside monophosphate added to the proviral transcript, and attaches it to either a free pyrophosphate (regenerating a deoxynucleoside triphosphate) or to a nucleoside di- or triphosphate (usually ATP). Uncoupling a chain-terminating NRTI residue therefore rescues reverse transcription and reduces drug susceptibility across the class, since the process is not specific for the selecting drug. Of all the nucleoside-associated mutations, the best known and most studied are the six associated with thymidine analogue exposure. These six mutations (M41L, D67N, K70R, L210W, T215Y/F, K219Q) enhance RT pyrophosphorolysis to confer high-level viral resistance to zidovudine, and clinically significant loss of response to stavudine and didanosine. They have also been found to confer reduced susceptibility to lamivudine and abacavir, particularly when present alongside other NRTI-induced changes. Other key mutations generally confer more limited resistance to specific agents, although the primary lamivudine- and abacavir-associated M184V substitution generates a broad spectrum of drug-dependent phenotypes, and uncommon mutational complexes conferring resistance across the entire class are well known. In addition to 'classical' multi-nucleoside-resistant genotypes, database-driven 'virtual phenotyping' for accumulations of NRTI-associated mutations around a core of thymidine analogue-induced changes predicts drug susceptibilities below wild-type across the entire NRTI class, even in the absence of key mutations associated with individual agents. When the natural range of drug susceptibilities for treatment-naive isolates is used as the basis for defining resistance, retrospective analysis of clinical isolates in the Virco database shows a significantly increased incidence of reduced susceptibility for the dideoxy NRTIs (didanosine, stavudine and zalcitabine) that was undetected in previous assays. These data imply a cumulative degradation of response to

Anti-HIV Agents↗
PubMed2001

Mutations in pea seedborne mosaic virus genome-linked protein VPg after pathotype-specific virulence in Pisum sativum.

Pisum sativum plant introduction (PI) line 269818 is resistant to potyvirus pea seedborne mosaic virus (PSbMV) isolates, categorized as pathotype P1, and is susceptible to pathotype P4 isolates. This difference in infectivity is determined by the viral genome-linked protein (VPg) cistron. Mutational analysis of VPg of PSbMV isolates DPD1 and NY representing pathotypes P1 and P4 revealed that codon changes affecting amino acids 105 to 117 in the central region of VPg influenced virulence on PI 269818. In contrast, infectivity on pea cultivar Dark Skinned Perfection, which is susceptible to both pathotypes, was not affected by the mutations. Mutants overcoming resistance in PI 269818 were analyzed for changes in the VPg coding region upon passage through PI 269818 and Dark Skinned Perfection. Adaptive changes were observed only upon passage through PI 269818 and only at codons from amino acid 105 to 117. Expression of DPD1 VPg in PI 269818 did not affect infection by NY, which suggests that VPg from DPD1 is not an elicitor of a general resistance response. The results are compatible with the hypothesis that viral amplification depends upon the interaction between VPg and a host factor.

Amino Acid Sequence↗
PubMed1991

Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA primer bound near the 5' end of the genomic RNA at a position termed the primer-binding site (PBS). The PBS is an 18-nucleotide region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2000

Methods in clinical molecular genetics.

UNLABELLED: Mutation detection is fundamental throughout biology, but especially so in medicine and medical research, particularly in gene discovery and cancer diagnostics. Current methods are unsatisfactory due to a range of reasons, but particularly due to cost and lack of convenience. The current methods are reviewed and their advantages and disadvantages discussed. The high profile chip technology whilst making an enormous impact in expression studies is more problematic for mutation detection, especially for unknown mutations and the problems are discussed. Finally, once mutations are discovered they need documenting accurately and the efforts of the Human Genome Organisation mutation database initiative due this direction are discussed. CONCLUSION: Current methods of mutation analysis, although unsatisfactory, are reviewed and the impact of new technologies assessed.

Databases, Factual↗
PubMed2002

Microsatellite instability and its relevance to cutaneous tumorigenesis.

Increasing evidence suggests that human tumors sequentially accumulate multiple mutations that cannot be explained by the low rates of spontaneous mutations in normal cells (2-3 mutations/cell). The mathematical models estimate that for the solid tumors to develop, as many as 6-12 mutations are required in each tumor cell. Therefore, to account for such high mutation rates, it is proposed that tumor cells are genetically unstable, i.e. they have genome-wide mutations at short repetitive DNA sequences called microsatellites. Microsatellite repeats are scattered throughout the human genome, primarily in the non-coding regions, and can give rise to variants with increased or reduced lengths, i.e. microsatellite instability (MSI). This instability has been reported in an increasing number of cutaneous tumors including: melanocytic tumors, basal cell carcinomas and primary cutaneous T-cell lymphomas. Moreover, MSI has been observed in skin tumors arising in the context of some hereditary disorders such as Muir-Torre syndrome, Von Recklinghausen's disease and disseminated superficial porokeratosis. While MSI in some of these disorders reflects underlying DNA replication errors, the mechanism of instability in others is still unknown. Thus far, MSI is considered to be a distinct tumorigenic pathway that reveals surprising versatility. The ramifications for cutaneous neoplasms warrant further investigation.

Animals↗
PubMed1997

BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients.

To date, more than 300 distinct small deletions, insertions and point mutations, mostly leading to premature termination of translation, have been reported in the breast/ovarian-cancer susceptibility gene BRCA1. The elevated frequencies of some mutations in certain ethnic subpopulations are caused by founder effects, rather than by mutation hotspots. Here we report that the currently available mutation spectrum of BRCA1 has been biased by PCR-based mutation-screening methods, such as SSCP, the protein truncation test (PTT) and direct sequencing, using genomic DNA as template. Three large genomic deletions that are not detected by these approaches comprise 36% of all BRCA1 mutations found in Dutch breast-cancer families to date. A 510-bp Alu-mediated deletion comprising exon 22 was found in 8 of 170 breast-cancer families recruited for research purposes and in 6 of 49 probands referred to the Amsterdam Family Cancer Clinic for genetic counselling. In addition, a 3,835-bp Alu-mediated deletion encompassing exon 13 was detected in 4 of 170 research families, while an deletion of approximately 14 kb was detected in a single family [corrected]. Haplotype analyses indicated that each recurrent deletion had a single common ancestor.

BRCA1 Protein↗
PubMed1996

In vivo assay of p53 function in homologous recombination between simian virus 40 chromosomes.

To investigate a possible role of p53 in DNA exchange mechanisms, we have developed a model system which allows us to quantify homologous recombination rates in eukaryotic cells. We generated two types of simian virus 40 (SV40) whose genomes were mutated in such a way that upon double infection of monkey cells, virus particles can be released only after interchromosomal exchange of genetic material. This test system allowed us to determine recombination rates in the order of 10(-4) to 10(-6) for chromatin-associated SV40 genomes. To study the role of p53-T-antigen (T-Ag) complexes in this process, we designed viral test genomes with an additional mutation leading to a single amino acid exchange in T-Ag (D402H) and specifically blocking T-Ag-p53 interactions. Analysis of primary rhesus monkey cells endogenously expressing wild-type p53 showed a decreased recombination rate upon loss of efficient T-Ag-p53 complex formation. However, cells expressing mutant p53 (LLC-MK2 cells), the introduction of mutant T-Ag did not affect the DNA exchange rates. Our data are interpreted to indicate an inhibitory role of wild-type p53 in recombination. In agreement with this hypothesis, p53-T-Ag complex formation alleviates the inhibitory effect of wild-type p53.

Animals↗
PubMed2006

Molecular mechanisms of poliovirus variation and evolution.

Replication of poliovirus RNA is accomplished by the error-prone viral RNA-dependent RNA polymerase and hence is accompanied by numerous mutations. In addition, genetic errors may be introduced by nonreplicative mechanisms. Resulting variability is manifested by point mutations and genomic rearrangements (e.g., deletions, insertions and recombination). After description of basic mechanisms underlying this variability, the review focuses on regularities of poliovirus evolution (mutation fixation) in tissue cultures, human organisms and populations.

Animals↗
PubMed2024

Critically unwell infants and children with mitochondrial disorders diagnosed by ultrarapid genomic sequencing.

PURPOSE: To characterize the diagnostic and clinical outcomes of a cohort of critically ill infants and children with suspected mitochondrial disorders (MD) undergoing ultrarapid genomic testing as part of a national program. METHODS: Ultrarapid genomic sequencing was performed in 454 families (genome sequencing: n = 290, exome sequencing +/- mitochondrial DNA sequencing: n = 164). In 91 individuals, MD was considered, prompting analysis using an MD virtual gene panel. These individuals were reviewed retrospectively and scored according to modified Nijmegen Mitochondrial Disease Criteria. RESULTS: A diagnosis was achieved in 47% (43/91) of individuals, 40% (17/43) of whom had an MD. Seven additional individuals in whom an MD was not suspected were diagnosed with an MD after broader analysis. Gene-agnostic analysis led to the discovery of 2 novel disease genes, with pathogenicity validated through targeted functional studies (CRLS1 and MRPL39). Functional studies enabled diagnosis in another 4 individuals. Of the 24 individuals ultimately diagnosed with an MD, 79% had a change in management, which included 53% whose care was redirected to palliation. CONCLUSION: Ultrarapid genetic diagnosis of MD in acutely unwell infants and children is critical for guiding decisions about the need for additional investigations and clinical management.

Humans↗
PubMed2001

DNA mismatch repair and cancer.

Five human DNA mismatch repair genes have been identified that, when mutated, cause susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Mutational inactivation of both copies of a DNA mismatch repair gene results in a profound repair defect and progressive accumulation of mutations throughout the genome. Some of the mutations confer selective advantage on the cells, giving rise to cancer. Recent discoveries suggest that apart from postreplication repair, DNA mismatch repair proteins have several other functions that are highly relevant to carcinogenesis. These include DNA damage surveillance, prevention of recombination between nonidentical sequences and participation in meiotic processes (chromosome pairing). A brief overview of these different features of the human DNA mismatch repair system will be provided, with the emphasis in their implications in cancer development.

Animals↗
PubMed1993

Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy.

Wild-type reverse transcriptase has evolved for the survival of human immunodeficiency virus type 1 (HIV-1) by natural selection. In contrast, therapy relying on inhibitors of reverse transcriptase by nucleosides like zidovudine (AZT) or dideoxyinosine (ddI), and by non-nucleosides like pyridinones or nevirapine, may exert different selection pressures on this enzyme. Therefore the acquisition of resistance to reverse transcriptase inhibitors by selection of mutations in the pol gene may require compromises in enzyme function that affect viral replication. As single mutations are unlikely to confer broad resistance when combinations of reverse transcriptase inhibitors are used, multiple mutations may occur that result in further compromises. Certain drug combinations may prevent the co-existence of adequate reverse transcription function and multi-drug resistance (MDR). Unlike bacterial or eukaryotic drug resistance, retroviral drug resistance is conferred only by mutations in its own genome and is limited by genome size. Combining drugs directed against the same essential viral protein may thus prevent HIV-1 MDR, whereas the conventional approach of targeting different HIV-1 proteins for combination therapy may not, because genomes with resistance mutations in different HIV-1 genes might recombine to develop MDR. Here we show that several mutations in the HIV-1 reverse transcriptase gene that confer resistance to inhibitors of this enzyme can attenuate viral replication. We tested whether combinations of mutations giving rise to single-agent resistance might further compromise or even abolish viral replication, and if multidrug-resistant viruses could be constructed. Certain combinations of mutations conferring resistance to AZT, ddI and pyridinone are incompatible with viral replication. These results indicate that evolutionary limitations exist to restrict development of MDR. Furthermore, a therapeutic strategy exploiting these limitations by using selected multidrug regimens directed against the same target may prevent development of MDR. This approach, which we call convergent combination therapy, eliminated HIV-1 replication and virus breakthrough in vitro, and may be applicable to other viral targets. Moreover, elimination of reverse transcription by convergent combination therapy may also limit MDR.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases.

We have explored the application of the bacteriophage resolvases T4 endonuclease VII and T7 endonuclease I for detecting mutations in genomic DNA. Heteroduplex DNA fragments prepared by amplification from DNA containing known mutations were cleaved by one or both enzymes at nucleotide mismatches created by 3 of 3 short deletions and 13 of 14 point mutations in fragments as large as 940 basepairs. Heteroduplexes representing all four classes of possible single nucleotide mismatches were cleaved, and the sizes of the cleavage products generated correlated with the location of the mutation. We conclude that bacteriophage resolvases may be useful reagents for the rapid screening of DNA for mutations.

Bacteriophages↗