DNA pooling in mutation detection with reference to sequence analysis

We discuss pooling methods of mutation detection for identifying rare mutations. We provide mathematical formulae for obtaining the optimal pool size as a function of the mutation frequency in the study population and the specificity of the test. The optimal pool size depends strongly on the specificity of the test. With a test that has 99% specificity, pooling can reduce the number of tests that need to be performed by 80%, whereas, with a test with 95% specificity, pooling reduces the number of samples that must be tested by only 50%. We used the software PHRED to call mutations after sequencing of pooled samples with known STK11 mutations. We found that, when the area under the curve for the less prominent peak was used to call mutations, we were able to pool pairs of samples and correctly identify mutations. Pooling of three samples did not lead to an adequately specific test for the basic automated allele-calling procedures that we used. We discuss methods by which the specificity may be improved to permit pooling of three or more samples when testing for mutations by sequencing.     

Biological significance of molecular chirality in energy balance and metabolism

Summary In biological electron transport the spin, and thus the magnetic property of electrons, is neglected. Furthermore, no attention is paid to the fact that the great majority of biologically important molecules are chiral, and during excitation a magnetic moment is induced in them. It is shown, both theoretically and experimentally, that the magnetic moment of the electron and the magnetic transition moment of the optically active molecules may interact. The main consequences of such an interaction are a higher probability of the occurrence of optically active molecules in triplet states, and the polarization of transported electrons.Note: The term chirality has been introduced byKelvin (Robert Boyle Lecture May 16, 1893, printed in Baltimore Lectures Appendix H p. 439, 1904). He wrote: I call any geometrical figure, or any group of points chiral, and say it has chirality, if its image in a plane mirror can not be brought to coincide with itself.     

Pseudogenes, junk DNA, and the dynamics of Rickettsia genomes

Studies of neutrally evolving sequences suggest that differences in eukaryotic genome sizes result from different rates of DNA loss. However, very few pseudogenes have been identified in microbial species, and the processes whereby genes and genomes deteriorate in bacteria remain largely unresolved. The typhus-causing agent, Rickettsia prowazekii, is exceptional in that as much as 24% of its 1.1-Mb genome consists of noncoding DNA and pseudogenes. To test the hypothesis that the noncoding DNA in the R. prowazekii genome represents degraded remnants of ancestral genes, we systematically examined all of the identified pseudogenes and their flanking sequences in three additional Rickettsia species. Consistent with the hypothesis, we observe sequence similarities between genes and pseudogenes in one species and intergenic DNA in another species. We show that the frequencies and average sizes of deletions are larger than insertions in neutrally evolving pseudogene sequences. Our results suggest that inactivated genetic material in the Rickettsia genomes deteriorates spontaneously due to a mutation bias for deletions and that the noncoding sequences represent DNA in the final stages of this degenerative process.     

A brief history of the status of transposable elements: from junk DNA to major players in evolution

The idea that some genetic factors are able to move around chromosomes emerged more than 60 years ago when Barbara McClintock first suggested that such elements existed and had a major role in controlling gene expression and that they also have had a major influence in reshaping genomes in evolution. It was many years, however, before the accumulation of data and theories showed that this latter revolutionary idea was correct although, understandably, it fell far short of our present view of the significant influence of what are now known as "transposable elements" in evolution. In this article, I summarize the main events that influenced my thinking about transposable elements as a young scientist and the influence and role of these specific genomic elements in evolution over subsequent years. Today, we recognize that the findings about genomic changes affected by transposable elements have considerably altered our view of the ways in which genomes evolve and work.     

Genome deterioration: loss of repeated sequences and accumulation of junk DNA

A global survey of microbial genomes reveals a correlation between genome size, repeat content and lifestyle. Free-living bacteria have large genomes with a high content of repeated sequences and self-propagating DNA, such as transposons and bacteriophages. In contrast, obligate intracellular bacteria have small genomes with a low content of repeated sequences and no or few genetic parasites. In extreme cases, such as in the 650kb-genomes of aphid endosymbionts of the genus Buchnera all repeated sequences above 200bp have been eliminated. We speculate that the initial downsizing of the genomes of obligate symbionts and parasites occurred by homologous recombination at repeated genes, leading to the loss of large blocks of DNA as well as to the consumption of repeated sequences. Further sequence elimination in these small genomes seems primarily to result from the accumulation of short deletions within genic sequences. This process may lead to temporary increases in the genomic content of pseudogenes and junk DNA. We discuss causes and long-term consequences of extreme genome size reductions in obligate intracellular bacteria.     

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.     

Exploring the common molecular basis for the universal DNA mutation bias: Revival of Löwdin mutation model

Recently, numerous genome analyses revealed the existence of a universal G:C → A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Löwdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Löwdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.     

Current understanding of UV-induced base pair substitution mutation in E. coli with particular reference to the DNA polymerase III complex

UV mutagenesis in E. coli is believed to occur in two discrete steps. The second step involves continued DNA synthesis beyond a blocking lesion in the template strand. This bypass step requires induced levels of umuD and umuC gene products and activated recA protein. DNA polymerase III may be involved since a dnaE mutator strain (believed to have defective base selection) is associated with enhanced UV mutagenesis in conjunction with a genetic background permitting the bypass step. In non-UV-mutable umu and lexA strains, UV mutagenesis can be demonstrated if delayed photorevesal is given. This is interpreted as indicating that an earlier misincorporation step can occur in such strains but the resulting mutations do not survive because the bypass step is blocked. The misincorporation step does not require any induced SOS gene products and can occur either at the replication fork or during repair replication following excision of a DNA lesion. Neither a dnaE mutator gene (leading to a defective subunit of DNA polymerase III holoenzyme) nor a mutD5 mutator gene (leading to a defective ε proofreading subunit) had any effect on he misincorporation step. Although this is consistent with DNA polymerase III holoenzyme not being involved in the misincorporation step, other interpretations involving the inhibition of ε proofreading activity by recA protein are possible.

In vitro studies are reported in which sites of termination of synthesis by DNA polymerase III holoenzyme on UV-irradiated M13 mp8 DNA were examined in the presence of inhibitors of the 3′–5′ proofreading exonuclease (including recA protein). No evidence was found for incorporation of bases opposite photoproducts suggesting that either inhibition is more complete in the cell and/or that other factors are involved in the misincorporation step.     

Mammalian DNA replication: mutation biases and the mutation rate

Experimental studies have shown that the fidelity of DNA replication can be affected by the concentrations of free deoxyribonucleotides present in the cell. Replication of mammalian chromosomes is achieved using pools of newly-synthesized deoxyribonucleotides which fluctuate during the cell cycle. Since regions of mammalian chromosomes are replicated sequentially, there is the potential for differences among mammalian loci in both the relative and absolute frequencies of the various transitional and transversional mutations which may occur. Where these mutations are effectively neutral, at silent sites in genes and in non-coding sequences, this may result in different rates of evolution and in different base compositions, as have been observed in data from mammalian genes. A simple model of the DNA replication process is developed to describe how the mutation rate could be affected by the G + C contents of the deoxyribonucleotide pools and of the replicating DNA. Mutation rates are predicted to vary from locus to locus; only in the particular case of identical G + C contents in the DNA locus and the deoxyribonucleotide pools, and no proofreading, will the mutation rate be uniform over all loci.     

Biological significance of the pseudomycelium in asporogenous yeasts

Summary By means of a quantitative investigation of mycelium formation in an asporogenous yeast the author reached the conclusion that pseudomycelium-production facilitates the absorption of nutrients when these are highly diluted in the culture medium. The reciprocical inhibition of mycelia by parallel growing colonies does not depend upon an inhibitory substance diffusing into the medium, but on the exhaustion of the culture medium.

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Résumé L'auteur ayant analizé par rechèrches quantitatives le phénomène de la filamentisation dans un champignon levuriforme conclue que la filamentisation a le but de faciliter l'absorption des matérieux nutritifs lorsque ces-ci sont bien dilués dans le milieu de culture. L'inibition réciproque entre le pseudomycelia des colonies de levure parallèles n'est pas déterminé par une substance emp/'echante qui se trasmet dans le milieu de culture, mais seulement par un empauvrissement des matérieux nutritifs.

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Riassunto I'A., avendo analizzato con esperienze quantitative, il fenomeno della filamentizzazione in un lievito asporigeno conclude che la comparsa del filamento ha lo scopo di facilitare l'assorbimento dei materiali nutritivi quando questi sono troppo scarsi nel mezzo di cultura. L'inibizione reciproca dei filamenti di colonie di lievito parallele non è dovuta ad una sostanza inibitrice diffusa nel mezzo di cultura ma semplicemente ad un impoverimento del mezzo culturale stesso.

     

Endogenous DNA damage and mutation

In humans, approximately 10(7) cells divide per second. Estimates suggest that spontaneous mutations arise in about a third of those cells. These mutations arise as mistakes in DNA replication and when DNA polymerases copy damaged templates. The latter result from chemical hydrolysis of nucleoside bases or by reaction of DNA with electrophiles or reactive free radicals generated during metabolism (endogenous DNA damaging agents). This article highlights recent discoveries and emerging opportunities in the study of endogenous DNA damage and mutation.     

Effect of G40R mutation on the binding of human SRY protein to DNA: a molecular dynamics view

Molecular dynamics simulation was conducted to investigate the reason why the mutant G40R of hSRY protein has a low affinity for DNA. Compared with the previous dynamics results of the wild-type hSRY-HMG-DNA complex, the results of molecular dynamics simulation on the mutant G40R hSRY-HMG-DNA system demonstrated that the whole structure of DNA (especially the second strand) had a major deviation away from the short arm of the HMG box. Consequently, the DNA and the mutant protein could not specifically recognize each other, that is, very different, and low-occupancy, direct, and water-mediated hydrogen bonds were detected at the protein-DNA interface, no conformational changes occurred at the loop region around Met9 during the simulation, and residue IIe13 did not intercalate between the bases of A5 and A6. These results indicated that the mutant G40R did not form a specific complex with the DNA target, hence led to complete gonadal dysgenesis. From the simulation, we realized that the residue Gly40 played a critical structural role in the hSRY-DNA recognition. It might be a structural supporting point of DNA binding because of the absence of a side chain. The reason for the difficulty of the mutant G40R to form a complex with DNA might be that the long and positively charged side chain of Arg40 by its bulk and positive charge hindered the DNA's access to the active sites of the protein.     

Biological horizons in molecular microscopy.

The purpose envisaged in this report is not to provide a comprehensive monography but rather to give a survey, especially for biologists, of the state of the art and of current research trends in molecular microscopy. Following a brief discussion of the obvious discrepancy between instrumental capabilities and the limits of biologically significant information, a definition of the diversifying field is attempted. Four main topics are discussed. First, recent progress in the field of "low noise" specimen supports is reviewed. It is emphasized that a minimum background structure is an important but not the sole criterion for a satisfactory support. It is the ability to adsorb molecules in a predeterminable and orderly fashion which will attract wider attention in the future; positional and orientational order figure as crucial points in the strategem of low dose microscopy. Second, the problem of achieving adequate contrast without the expense of an unfaithful representation of molecular structures is discussed. Contrast is a problem of optimum imaging modes as well as of preparatory techniques. The third topic of discussion is specimen dehydration. Several avenues to circumvent or at least to alleviate dehydration artifacts are outlined. The last chapter focusses on the most fundamental problem in molecular microscopy:radiation damage. A brief synopsis of the physical and physico-chemical processes involved in damaging interactions is given and an attempt is made to tesselate the true picture of radiation damage to lipids and proteins. This might serve as a guidance in assessing the degree of structural fidelity to be expected for a given electron dose. Possibilities to overcome the radiation damage problem are adumbrated.