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PubMed2000

Estimation of parameters of deleterious mutations in partial selfing or partial outcrossing populations and in nonequilibrium populations.

The Deng-Lynch method was developed to estimate the rate and effects of deleterious genomic mutations (DGM) in natural populations under the assumption that populations are either completely outcrossing or completely selfing and that populations are at mutation-selection (M-S) balance. However, in many plant and animal populations, selfing or outcrossing is often incomplete in that a proportion of populations undergo inbreeding while the rest are outcrossing. In addition, the degrees of deviation of populations from M-S balance are often not known. Through computer simulations, we investigated the robustness and the applicability of the Deng-Lynch method under different degrees of partial selfing or partial outcrossing and for nonequilibrium populations approaching M-S balance at different stages. The investigation was implemented under constant, variable, and epistatic mutation effects. We found that, generally, the estimation by the Deng-Lynch method is fairly robust if the selfing rate (S) is <0.10 in outcrossing populations and if S > 0.8 in selfing populations. The estimation may be unbiased under partial selfing with variable and epistatic mutation effects in predominantly outcrossing populations. The estimation is fairly robust in nonequilibrium populations at different stages approaching M-S balance. The dynamics of populations approaching M-S balance under various parameters are also studied. Under mutation and selection, populations approach balance at a rapid pace. Generally, it takes 400-2000 generations to reach M-S balance even when starting from homogeneous individuals free of DGM. Our investigation here provides a basis for characterizing DGM in partial selfing or outcrossing populations and for nonequilibrium populations.

Genetics, Population↗
PubMed2006

Extended gene analysis can increase specificity of neonatal screening for cystic fibrosis.

AIM: To assess whether carriers and patients can be accurately identified by extended gene analysis for cystic fibrosis (CF) in dried blood spots. METHODS: A blinded analysis was performed in 10-mm2 blood spots on Guthrie cards, punched as if to remove material for the IRT test, from 10 CF patients and 10 carriers with known CF mutations. Genomic DNA was isolated. Aliquots of 1 microl dissolved DNA were used for subsequent PCRs. Analysis of the deltaF508 mutation was followed by an oligonucleotide ligation assay. Denaturing gradient gel electrophoresis of the whole CFTR gene was carried out in samples with only one identified mutation. Amplicons revealing an aberrant pattern were sequenced. RESULTS: In all cases, the blood-spot genotype was identical to that previously determined from whole-blood analysis. Estimated time needed to complete the procedure in a series of Guthrie cards was 3-4 wk. CONCLUSION: Extended gene analysis in dried blood spots can discriminate CF patients and carriers. If proven equally reliable in larger series, an approach to neonatal screening in which tests are only considered as screen positive when two CF mutations are found is possible. This can increase the specificity of the screening programme, and carrier detection can practically be avoided.

Algorithms↗
PubMed2002

Mobile genetic element activation and genotoxic cancer therapy: potential clinical implications.

Approximately one-quarter of the human genome is composed of short and long interspersed elements (SINEs and LINEs, respectively). These elements have spread throughout the genome by a process termed retrotransposition, consisting of transcription of an element into RNA, reverse transcription into cDNA, and reinsertion of the copied element into a new genomic location. Recombination events involving these elements, including novel insertions into active genes, have been associated with a number of human diseases. Despite the fact that these elements have replicated to hundreds of thousands of copies in the genome, under most conditions they remain transcriptionally silent, and therefore are not actively replicating. The signals controlling retrotransposable element activation in the genome have not been defined. Our laboratory recently found that exposure of cells to a variety of DNA-damaging agents, including several common chemotherapeutic drugs and gamma-radiation, is associated with dramatic induction of SINE transcription, and of a concomitant endogenous reverse transcriptase activity. As SINEs do not encode for reverse transcriptase, the latter finding suggests a more global activation of retrotransposable elements in response to DNA damage. Together these observations suggest that genotoxic exposure may lead to genomic mutation not only through direct DNA damage, but also through indirect activation of potentially mutagenic mobile elements in the genome. The nonrandom distribution of retrotransposable elements in the human genome may contribute to the pattern of characteristic translocation events associated with secondary malignancies in patients exposed to genotoxic agents. Here we describe these and other mechanisms by which retrotransposable elements can contribute to disease, and present an overview of what is known about this large, and largely unexplored, segment of the genome. Understanding the cellular responses to genotoxic stress may permit the development of a means of predicting the risks and preventing the development of secondary malignancy following cancer therapy.

Antineoplastic Agents↗
PubMed1999

Hepatitis B virus variants with lamivudine-related mutations in the DNA polymerase and the 'a' epitope of the surface antigen are sensitive to ganciclovir.

Lamivudine is a new antiviral agent effective against hepatitis B viral (HBV) infections but can result in virus-drug resistance associated with mutations in the conserved 'YM552DD' motif of the HBV DNA polymerase. Due to their overlapping coding regions in the HBV genome, mutations in the DNA polymerase may result in substitutions in the hepatitis B surface antigen (HBsAg), albeit outside the antigenic 'a' epitope. Here we report the identification of a novel type of lamivudine-related mutations located in both the polymerase (YM552DD-->Y1552DD) and the 'a' epitope of HBsAg (Gly130-->Asp130). The same virus carried a HBsAg Gly145-->Arg145 mutation prior to therapy. Both the wild type HBV and lamivudine-related mutants with the Gly145-->Arg145 HBsAg mutation were suppressed following ganciclovir treatment, indicating a beneficial additive effect of both drugs against different forms of HBV mutants.

Adult↗
PubMed1997

The yeast Saccharomyces cerevisiae as a genetic system for obtaining variants of poliovirus protease 2A.

The inducible expression of poliovirus protease 2A (2Apro) blocks the growth of Saccharomyces cerevisiae. A number of yeast colonies that grow after 2Apro induction have been isolated. The majority of these clones express 2Apro to control levels, suggesting that their ability to divide is not due to the loss of 2Apro gene inducibility. The sequences of the 2Apro genes isolated from 22 clones were determined. Most of the 2Apro sequences from these colonies contain point mutations in the poliovirus protease. The different variant protease sequences were transferred to an infectious poliovirus cDNA clone. Translation of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cleave P1-2A in cis. In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently. Transfection of the RNA generated from the full-length poliovirus genomes mutated in 2Apro yielded five viable polioviruses with a small plaque phenotype. These five polioviruses efficiently cleaved p220 but showed defects in viral protein synthesis, transactivation of a leader-luciferase mRNA, and 3CD cleavage to 3C' and 3D'. All 2Apro mutant sequences, including those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter. Only the 2Apro variants that have activity to cleave 3CD produced viable poliovirus. Our findings indicate that S. cerevisiae represents a useful system for obtaining poliovirus 2Apro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.

Amino Acid Sequence↗
PubMed1991

Spontaneous mutation at the hypervariable mouse minisatellite locus Ms6-hm: flanking DNA sequence and analysis of germline and early somatic mutation events.

Hypervariability at minisatellite loci is maintained by spontaneous mutation to new-length alleles. At the most variable loci, mutation rate is directly measurable by pedigree analysis. The mouse minisatellite locus Ms6-hm has a germline mutation rate of 2.5% per gamete and is therefore one of the most unstable loci yet identified in the mouse genome. Mutation events at this locus also occur during early mouse development, resulting in mice mosaic for cells carrying a common non-parental allele in different somatic tissues and the germline. The DNA sequence flanking Ms6-hm is rich in dispersed repetitive elements; the minisatellite array has expanded from within a member of the Mouse Transcript family which is flanked by two additional Mouse Transcript elements, and a B2 element lies further 3' to the minisatellite. To define the characteristics of mutation events at Ms6-hm we have analysed 19 germline and 13 somatic length-change events. Germline mutation events at Ms6-hm are not accompanied by the exchange of flanking markers in three informative mutant alleles analysed.

Alleles↗
PubMed1993

Myoclonus epilepsy and ragged-red fibers: blood mitochondrial DNA heteroplasmy in affected and asymptomatic members of a family.

By a rapid PCR-based method to assess the 8344 mtDNA mutation associated with MERRF disease, we have studied DNA from blood samples of 10 individuals belonging to a family spanning four generations in which one patient showed the complete MERRF phenotype, three other members were less severely affected, while the remaining were unaffected. The percentage of mutant mtDNA was quantified by laser-densitometric scanning of the negative photographic sheets of the agarose gels. The results showed that the MERRF patient had 53% of mutated mtDNA while the two less affected patients had 62% and 14% of mutated mtDNA, respectively. However, a high percentage of mutated genomes (up to 64%) was also found in some unaffected relatives. These results show that although on one hand the mutation is probably the primary cause of the disease, on the other hand the relative amount of mutated mtDNA in blood samples is not indicative of its clinical expression.

Adolescent↗
PubMed2006

Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication.

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

Amino Acid Sequence↗
PubMed2004

Aberrant and alternative splicing in cancer.

Pre-mRNA splicing is a sophisticated and ubiquitous nuclear process, which is a natural source of cancer-causing errors in gene expression. Intronic splice site mutations of tumor suppressor genes often cause exon-skipping events that truncate proteins just like classical nonsense mutations. Also, many studies over the last 20 years have reported cancer-specific alternative splicing in the absence of genomic mutations. Affected proteins include transcription factors, cell signal transducers, and components of the extracellular matrix. Antibodies against alternatively spliced products on cancer cells are currently in clinical trials, and competitive reverse transcription-PCR across regions of alternative splicing is being used as a simple diagnostic test. As well as being associated with cancer, the nature of the alternative gene products is usually consistent with an active role in cancer; therefore, the alternative splicing process itself is a potential target for gene therapy.

Alternative Splicing↗
PubMed1998

Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.

Binding Sites↗
PubMed2000

Kluyveromyces lactis Sir2p regulates cation sensitivity and maintains a specialized chromatin structure at the cryptic alpha-locus.

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating type loci requires the formation of a heterochromatin-like structure, which is dependent on silent information regulator (Sir) proteins and DNA sequences, called silencers. To learn more about silencing, we characterized the mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HMLalpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switching. HMRa contained two genes, the a1 gene similar to the Saccharomyces a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HMLalpha contained three genes, the alpha1 and alpha2 genes, which were similar to their Saccharomyces counterparts, and a novel third gene, alpha3. A dam-methylase assay showed Sir-dependent, but transcription-independent changes of the chromatin structure of the HMLalpha locus. The HMLalpha3 gene did not appear to be part of the silent domain because alpha3p was expressed from both MATalpha3 and HMLalpha3 and sir mutations failed to change the chromatin structure of the HMLalpha3 gene. Furthermore, a 102-bp silencer element was isolated from the HMLalpha flanking DNA. HMLalpha was also flanked by an autonomously replicating sequence (ARS) activity, but the ARS activity did not appear to be required for silencer function. K. lactis sir2 strains grown in the presence of ethidium bromide (EtBr) accumulated the drug, which interfered with the essential mitochondrial genome. Mutations that bypassed the requirement for the mitochondrial genome also bypassed the EtBr sensitivity of sir2 strains. Sir2p localized to the nucleus, indicating that the role of Sir2p to hinder EtBr accumulation was an indirect regulatory effect. Sir2p was also required for growth in the presence of high concentrations of Ni(2+) and Cu(2+).

Base Sequence↗
PubMed1998

Evolutionarily stable mutation rates.

I investigate the hypothesis that mutation rates in natural populations are determined by a balance between: (1) selection against deleterious mutations favouring lower mutation rates, and (2) selection opposing further reduction of the mutation rate, resulting from the costs incurred by more stringent proof-reading and repair (for example, a reduction in the rate of DNA replication). The influence of advantageous mutations is assumed to be negligible. In a previous paper, I analysed the dynamics of a modifier of the mutation rate in a large sexual population, where (infinitesimally rare) deleterious alleles segregate at an infinite number of unlinked loci with symmetric multiplicative fitness effects. A simple condition was obtained for a modifier allele to increase in frequency. Remarkably, this condition does not depend on the allele frequencies at the modifier locus. Here, I show that (as a consequence), given any set of possible values of the mutation rate (any set of possible modifier alleles), there always exists a single globally stable value of the mutation rate. This is an unusually strong form of "evolutionary stability" for a sexual population. Less surprisingly the optimum mutation rate in an asexual population has similar stability properties. Furthermore, in the case of an asexual population, it is not necessary to make any special assumptions about the selection acting against deleterious mutations, except that a deterministic mutation-selection equilibrium exists. I present a simple method for identifying the evolutionarily stable value of the mutation rate, given the function alpha(U) relating the value of the mutation rate to the fitness cost of maintaining this rate. I also argue that if there is a highly conserved relationship between the rate of replication per base, and the rate of mutation per base, and if this relationship has the form of a power law, then the remarkable uniformity of the per genome mutation rate in DNA based microbes can be explained.

Animals↗
PubMed1996

Estimation of deleterious-mutation parameters in natural populations.

The rate and average effects of spontaneous deleterious mutations are important determinants of the evolution of breeding systems and of the vulnerability of small populations to extinction. Nevertheless, few attempts have been made to estimate the properties of such mutations, and those studies that have been performed have been extremely labor intensive, relying on long-term, laboratory mutation-accumulation experiments. We present an alternative to the latter approach. For populations in which the genetic variance for fitness is a consequence of selection-mutation balance, the mean fitness and genetic variance of fitness in outbred generations can be expressed as simple functions of the genomic mutation rate, average homozygous effect and average dominance coefficient of new mutations. Using empirical estimates for the mean and genetic variance of fitness, these expressions can then be solved to obtain joint estimates of the deleterious-mutation parameters. We employ computer simulations to evaluate the degree of bias of the estimators and present some general recommendations on the application of the technique. Our procedures provide some hope for obtaining estimates of the properties of deleterious mutations from a wide phylogenetic range of species as well as a mechanism for testing the validity of alternative models for the maintenance of genetic variance for fitness.

Gene Deletion↗
PubMed2004

Prevalence of SAP gene defects in male patients diagnosed with common variable immunodeficiency.

The molecular basis of common variable immunodeficiency (CVID) is undefined, and diagnosis requires exclusion of other diseases including X-linked lymphoproliferative disease (XLP). This rare disorder of immunedysregulation presents typically after Epstein-Barr virus infection and results from defects in the SAP (SLAM associated protein) gene. SAP mutations have been found in a few patients diagnosed previously as CVID, suggesting that XLP may mimic CVID, but no large-scale analysis of CVID patients has been undertaken. We therefore analysed 60 male CVID and hypogammaglobulinaemic patients for abnormalities in SAP protein expression and for mutations in the SAP gene. In this study only one individual, who was found later to have an X-linked family history, was found to have a genomic mutation leading to abnormal SAP cDNA and protein expression. These results demonstrate that SAP defects are rarely observed in CVID patients. We suggest that routine screening of SAP may only be necessary in patients with other suggestive clinical features.

Adolescent↗
PubMed2001

Efficient detection of Alport syndrome COL4A5 mutations with multiplex genomic PCR-SSCP.

We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer condition and PCR profile. Application of the method to a consecutive series of 46 United States patients with diverse indications of Alport syndrome resulted in detection of mutations in 31 cases and of five previously unreported polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sensitivity is greater than 79%. The test examines 52 segments, including the COL4A6/COL4A5 intergenic promoter region, all 51 of the previously recognized exons and two newly detected exons between exons 41 and 42 that encode an alternatively spliced mRNA segment. New genomic sequence information was generated and used to design primer pairs that span substantial intron sequences on each side of all 53 exons. For SSCP screening, 16 multiplex PCR combinations (15 4-plex and 1 3-plex) were used to provide complete, partially redundant coverage of the gene. The selected combinations allow clear resolution of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missense change in exon 32 but was found to cause exon skipping. Another missense variant may mark a novel functional site located in the collagenous domain.

Base Sequence↗
PubMed2003

Determination of -3858G-->A and -164C-->A genetic polymorphisms of CYP1A2 in blood and saliva by rapid allelic discrimination: large difference in the prevalence of the -3858G-->A mutation between Caucasians and Asians.

INTRODUCTION: Two mutations in CYP1A2, -164C-->A (allele CYP1A2*F) and -3858G-->A (allele CYP1A2*C), affecting the inducibility of the enzyme, have been published. The aim of this study was to develop a high throughput allelic discrimination assay for these mutations in both saliva and blood and to determine their frequency in Caucasians. METHODS: An allelic discrimination assay, based on the fluorogenic 5'-nuclease activity (TaqMan), was developed for the two mutations. Genomic DNA extracted from 17 saliva and 100 blood samples from Caucasians was analysed. RESULTS AND CONCLUSIONS: For the -164C-->A mutation, we found an allelic frequency of 68% in the Caucasian population, comparable with data published for Asians and Caucasians. For the -3858G-->A mutation, the allele frequency was only 2% in Caucasians, a much lower value than the approximately 25% reported in Asians (P<0.001). The presented allelic discrimination allows fast and accurate detection of these two mutations. Genotype calls were 100% identical for DNA from saliva and blood. Saliva is easily accessible and represents an excellent alternative to the traditionally used venous blood for genotyping.

Asian People↗
PubMedDate not supplied

[Genetic and somatic effects of ionizing radiation in humans and animals (comparative aspect)].

Genetic effects of ionizing radiation in the progeny of exposed parents could be conventionally subdivided on three main types. 1. Severe developmental disorders (fetus death, stillbirth, early postnatal mortality, malformation, hereditary disease, sterility). These effects are known to be caused by so called "gross" mutations (genomic, chromosomal, those of essential genes) with dominant harmful effects. Although found in rodents, insects, fishes they have not been found in humans. The proposal is made that the higher reproductive potency of a species the higher its tolerance for defective organisms. Due to strong selection against severe defects at the earliest stage of pregnancy, this genetic effect of radiation is likely to be difficult to detect in people. 2. Increased cancer risk manifested as elevated incidence of spontaneous tumors and increased sensitivity to carcinogenic agents. 3. Decreased fitness (non-carcinogenic negative health effects). These two last types of radiation genetic effects are presumably due to instability and functional inferiority of cell genome in the progeny of irradiated parents. The genetic background of these effects is suggested to be the load of induced minor mutations in regulatory genes (mini-, microsatelite loci) or/and epigenomic rearrangements of DNA in parental germ cells transmitted to progeny. These radiation genetic effects are much more obvious in animals as compared to humans. Apparently, they are difficult to find in humans because of their essential dependence on promotive (life style) factors, which are impossible to control in the offspring of irradiated people. A comparison of somatic (in irradiated organisms) and genetic (in the progeny of irradiated parents) effects of radiation provide evidence on the phenomenological as well as pathogenic similarity.

Animals↗
PubMed2004

Information dynamics in carcinogenesis and tumor growth.

The storage and transmission of information is vital to the function of normal and transformed cells. We use methods from information theory and Monte Carlo theory to analyze the role of information in carcinogenesis. Our analysis demonstrates that, during somatic evolution of the malignant phenotype, the accumulation of genomic mutations degrades intracellular information. However, the degradation is constrained by the Darwinian somatic ecology in which mutant clones proliferate only when the mutation confers a selective growth advantage. In that environment, genes that normally decrease cellular proliferation, such as tumor suppressor or differentiation genes, suffer maximum information degradation. Conversely, those that increase proliferation, such as oncogenes, are conserved or exhibit only gain of function mutations. These constraints shield most cellular populations from catastrophic mutator-induced loss of the transmembrane entropy gradient and, therefore, cell death. The dynamics of constrained information degradation during carcinogenesis cause the tumor genome to asymptotically approach a minimum information state that is manifested clinically as dedifferentiation and unconstrained proliferation. Extreme physical information (EPI) theory demonstrates that altered information flow from cancer cells to their environment will manifest in-vivo as power law tumor growth with an exponent of size 1.62. This prediction is based only on the assumption that tumor cells are at an absolute information minimum and are capable of "free field" growth that is, they are unconstrained by external biological parameters. The prediction agrees remarkably well with several studies demonstrating power law growth in small human breast cancers with an exponent of 1.72+/-0.24. This successful derivation of an analytic expression for cancer growth from EPI alone supports the conceptual model that carcinogenesis is a process of constrained information degradation and that malignant cells are minimum information systems. EPI theory also predicts that the estimated age of a clinically observed tumor is subject to a root-mean square error of about 30%. This is due to information loss and tissue disorganization and probably manifests as a randomly variable lag phase in the growth pattern that has been observed experimentally. This difference between tumor size and age may impose a fundamental limit on the efficacy of screening based on early detection of small tumors. Independent of the EPI analysis, Monte Carlo methods are applied to predict statistical tumor growth due to perturbed information flow from the environment into transformed cells. A "simplest" Monte Carlo model is suggested by the findings in the EPI approach that tumor growth arises out of a minimally complex mechanism. The outputs of large numbers of simulations show that (a) about 40% of the populations do not survive the first two-generations due to mutations in critical gene segments; but (b) those that do survive will experience power law growth identical to the predicted rate obtained from the independent EPI approach. The agreement between these two very different approaches to the problem strongly supports the idea that tumor cells regress to a state of minimum information during carcinogenesis, and that information dynamics are integrally related to tumor development and growth.

Information Theory↗