Mapping the order of DNA restriction fragments

A straightforward method was designed for mapping the order of DNA restriction fragments obtained by a double and two single digestions, without the necessity of using a computer or a radioactive label. All possible solutions compatible with a pre-set level of error in the determination of sequence lengths are obtained. The primary assumptions are given, and the appropriate modifications of the algorithm are presented as a function of any assumptions one is unable (or unwilling) to make. Use of the method in connection with end-labeled fragments is also described.     

New equations and a method for finding nine parameter values for two alleles at one locus to study gene conversion using Ascobolus immersus.

A quantitative treatment is given for meiotic gene conversion with its parameters and equations for their interactions to determine allele segregation class frequencies from heterozygotes. The possible pairing of both pairs of nonsister chromatids in a bivalent at exactly the same point is included. Using sets of data from Ascobolus immersus, it is shown that values for all nine parameters for hybrid DNA models of recombination can be obtained using an iterative computer program. The accuracy of the values is estimated and the double-strand gap repair model is considered. The parameter values obtained invalidate most of the simplifications used in previous quantitative analyses of gene conversion data. They showed total bias in strand preference in asymmetric hybrid DNA formation and some bias in which type of chromatid is the invading one. There were slight differences in repair frequency between the two types of mispair and very large differences in the direction of repair. Conversion control factors had major effects on hybrid DNA formation and repair of mispairs.     

Gene Conversion at the Gray Locus of SORDARIA FIMICOLA: Fit of the Experimental Data to a Hybrid DNA Model of Recombination

A hybrid DNA (hDNA) model of recombination has been algebraically formulated, which allows the prediction of frequencies of postmeiotic segregation and conversion of a given allele and their probability of being associated with a crossing over. The model considered is essentially the "Aviemore model." In contrast to some other interpretations of recombination, it states that gene conversion can only result from the repair of heteroduplex hDNA, with postmeiotic segregation resulting from unrepaired heteroduplexes. The model also postulates that crossing over always occurs distally to the initiation site of the hDNA. Eleven types of conversion and postmeiotic segregation with or without associated crossover were considered. Their theoretical frequencies are given by 11 linear equations with ten variables, four describing heteroduplex repair, four giving the probability of hDNA formation and its topological properties and two giving the probability that crossing over occurs at the left or right of the converting allele. Using the experimental data of Kitani and coworkers on conversion at the six best studied gray alleles of Sordaria fimicola, we found that the model considered fit the data at a P level above or very close (allele h4) to the 5% level of sampling error provided that the hDNA is partly asymmetric. The best fitting solutions are such that the hDNA has an equal probability of being formed on either chromatid or, alternatively, that both DNA strands have the same probability of acting as the invading strand during hDNA formation. The two mismatches corresponding to a given allele are repaired with different efficiencies. Optimal solutions are found if one allows for repair to be more efficient on the asymmetric hDNA than on the symmetric one. In the case of allele g1, our data imply that the direction of repair is nonrandom with respect to the strand on which it occurs.     

Successes and prospects for genetic engineering]

The review of literature (1970-1976) on problems of gene engineering is given. Gene engineering is pointed out to be a new method of modern biology and a new page of modern molecular genetics. Gene engineering detected a real possibility of artificial creating living hybrid organisms, i.e. constructing functional recombinant DNA molecules according to a project of investigator, but not to possibilities of crossing. The determination of gene engineering (in contrast with genetical engineering) is given in the first division of the article. Genetical engineering is a construction of hybrid organisms on the basis of recombination between non-homologous chromosomes cy crossing. Genetical engineering is based on sex crossing, thus the application of this method is restricted by crossability (i.e. experiments in vivo), which possibilities are determined by taxonomical limits. Gene engineering is a new method of operating directly with genes. It permits constructing in vitro any hybrid genomes desirable. There is no limits of combining ability for gene engineering. Three main stages of constructing hybrid genomes should be taken into account for the proper determination of gene engineering as a method of genome constructing: 1) the gene isolation; 2) their cross-linking in vitro; 3) the transfer of hybrid DNA into recipient cell or its genome. The cardinal stage of gene engineering is the construction of hybrid DNA, cross-linking any initial DNAs from any remote animals, plants and bacteria. All the methods known of gene isolation are described. The chemical method of gene isolation is based on that case, when DNA of some gene differs in its physico-chemical characteristics from total DNA, for example, DNAs of genes coding ribosomal RNAs or sea urchine histone DNA. Isolation of promotors and operators using DNA dependent RNA polymerase, which recognizes promotors, repressor and operator DNA, should also be considered as the chemical method of gene isolation. Restrictase method, which is also well known, is convenuent because the restricts have long enough sticky ends, which is important for the following gene cross-linking. The method of total restriction, reported by Lederberg et al. and Debabov et al., is described. The phage method (in particular, Shimada method) is given, permitting the direct integration of lambda phage into a number of sites of Escherichia coli chromosome. Gene engineering method of gene isolation is mentioned, in particular, the data of Kameron et al. on hybrid phages carrying DNA ligase gene, and Clark a. Carbon on hybrid plasmids carrying triptophane and arabinose operons genes. These methods are called "shot gun". Methods of gene isolation from higher organisms are less developed. A method of gene isolation using so called colony hybridization (according to Grünstein and Hognes) is also given...     

CELL LOSS FROM EXPERIMENTAL TUMOURS

The rate of loss of cells from a tumour may be estimated by measuring the cell production rate and comparing this with the rate at which cells are observed to be added to the tumour volume. An attractive method of measuring cell production rate is by the simultaneous measurement of a thymidine-labelling index and the duration of the DNA synthetic period. A theoretical treatment of this method in exponential populations is given and the necessary assumptions are indicated. Estimates of cell loss have been made for a number of experimental tumours using available published data. It is shown that in some cases cell loss is an important, perhaps even dominant, factor determining tumour growth rate and the shape of tumour growth curves.     

Biochemical analysis of genetic recombination in eukaryotes.

Recent studies concerning molecular mechanisms of genetic recombination in eukaryotes are reviewed. Since many of these studies have focused on the testable predictions arising from the hybrid DNA theory of genetic recombination, this theory is summarised. Experiments to determine the time of meiotic crossing-over and the structure of the synaptonemal complex which facilitates meiotic crossing-over are described. Investigations of DNA nicking and repair events implicated in recombination are discussed. Properties of proteins which may facilitate hybrid DNA formation, and biochemical evidence for hybrid DNA formation are presented. Finally, a nuclease which has been implicated in gene conversion is described.     

A stochastic model for in vitro ageing II. A theory of marginotomy

A model for variation in the lifespan of mass cultures and clones of human diploid fibroblasts is analyzed. The model is based on Olovnikov's theory of marginotomy and the following four assumptions: The time between successive divisions of a given cell may be split into a portion of fixed length and a portion of random length; cells divide or lose the ability to divide independently of one another; there is a fixed probability that a cell may lose one of its important parts during DNA synthesis; there is a small but fixed probability that a cell may lose all its important parts and hence the ability to replicate further during DNA synthesis. Samples taken by Monte Carlo means at successive stages in the development of a hypothetical population are compared with clonal survival data derived experimentally. The fit between theoretical and experimental findings is within the range of variation inherent in the experimental method. Applications of the theory and suggestions for further experiments are given.     

Chromatin dynamics during cell cycle mediate conversion of DNA damage into chromatid breaks and affect formation of chromosomal aberrations: biological and clinical significance

The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid.     

Methylation perturbations in retroelements within the genome of a Mus interspecific hybrid correlate with double minute chromosome formation

A reduction in the DNA modification of cytosine methylation has been linked directly to chromosome rearrangements concomitant with retroelement amplification in several marsupial hybrid genomes. While phenotypes observed for interspecific eutherian hybrids are suggestive of methylation perturbations and retroelement instability, no link between retroelements, DNA methylation, and chromosome instability has yet been identified. Previous studies in eutherian hybrids, however, have been limited to a gross examination of methylation using methylation-sensitive restriction enzyme analysis or focused on single-copy genes and/or have avoided examination of repetitive DNA. Methylation changes and retroelements are proposed as mechanisms for double minute chromosome formation and oncogene amplification, both present in the genome of a Mus hybrid model, thus making it an ideal system to evaluate methylation status more closely. We have used the PCR-based methodologies methylation-sensitive amplicon subtraction (MS-AS) and methylation-sensitive representational difference analysis (MS-RDA) to detect differentially methylated sequences between three complex genomes and to isolate methylation perturbations in a Mus musculusxMus caroli hybrid. This novel application of MS-AS and MS-RDA resulted in the isolation of differentially methylated retroelements surrounding the locus on Chromosome 10 responsible for double minute chromosome formation within this interspecific eutherian hybrid.     

Tangle analysis of Xer recombination reveals only three solutions, all consistent with a single three-dimensional topological pathway

The product of Xer recombination at directly repeated psi sites on a circular unknotted DNA molecule is a right-hand four-noded catenane. Here, we use tangle equations to analyze the topological changes associated with Xer recombination at psi. This mathematical method allows computation of all possible topological pathways consistent with the experimental data. We give a rigorous mathematical proof that, under reasonable biological assumptions, there are only three solutions to the tangle equations. One of the solutions corresponds to a synaptic complex with antiparallel alignment of recombination core sites, the other two correspond to parallel alignment of cores. We show that all three solutions can be unified into a single three-dimensional model for Xer recombination. Thus the three distinct mathematical solutions do not necessarily represent distinct three-dimensional pathways, and in this case the three distinct tangle solutions are different planar projections of the same three-dimensional configuration.     

Domains for Protein-Protein Interactions at the N and C Termini of the Large Subunit of Bacteriophage λ Terminase

The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antarctic DNA barcoding; a drop in the ocean?

Coordinated, circum-Antarctic sampling expeditions during International Polar Year 2008/09 have given access to comprehensive collections suitable for DNA barcoding. Collaborations between the Census of Antarctic Marine Life (CAML), the Marine Barcode of Life project and the Canadian Centre for DNA Barcoding have enabled the Antarctic scientific community to initiate large-scale DNA barcoding projects to record the genetic diversity of Antarctic marine fauna, coordinated by the CAML Barcoding Campaign. A total of 20,355 marine specimens from more than 2,000 morphospecies covering 18 phyla are in the processing pipeline, and to date, 11,530 sequences have been processed with the remainder due by the end of 2010. Here, we present results on the current geographic and taxonomic coverage of DNA barcode data in the Southern Ocean and identify the remaining gaps. We show how DNA barcoding in the Antarctic is answering important questions regarding marine genetic diversity and challenging current assumptions of species distribution at the poles.     

Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses

Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.     

Normalization of microarray data: single-labeled and dual-labeled arrays

DNA microarray is a powerful tool for high-throughput analysis of biological systems. Various computational tools have been created to facilitate the analysis of the large volume of data produced in DNA microarray experiments. Normalization is a critical step for obtaining data that are reliable and usable for subsequent analysis such as identification of differentially expressed genes and clustering. A variety of normalization methods have been proposed over the past few years, but no methods are still perfect. Various assumptions are often taken in the process of normalization. Therefore, the knowledge of underlying assumption and principle of normalization would be helpful for the correct analysis of microarray data. We present a review of normalization techniques from single-labeled platforms such as the Affymetrix GeneChip array to dual-labeled platforms like spotted array focusing on their principles and assumptions.     

Optimal control of hybrid variable-order fractional coronavirus (2019-nCov) mathematical model; numerical treatments

A novel coronavirus is a serious global issue and has a negative impact on the economy of Egypt. According to the publicly reported data, the first case of the novel corona virus in Egypt was reported on 14 February 2020. Total of 96753 cases were recorded in Egypt from the beginning of the pandemic until the eighteenth of August, where 96, 581 individuals were Egyptians and 172 were foreigners. Recently, many mathematical models have been considered to better understand coronavirus infection. Most of these models are based on classical integer-order derivatives which can not capture the fading memory and crossover behavior found in many biological phenomena. Therefore, we study the coronavirus disease in this paper by exploring the dynamics of COVID-19 infection using new variable-order fractional derivatives. This paper presents an optimal control problem of the hybrid variable-order fractional model of Coronavirus. The variable-order fractional operator is modified by an auxiliary parameter in order to satisfy the dimensional matching between the both sides of the resultant variable-order fractional equations. Existence, uniqueness, boundedness, positivity, local and global stability of the solutions are proved. Two control variables are considered to reduce the transmission of infection into healthy people. To approximate the new hybrid variable-order operator, Grünwald-Letnikov approximation is used. Finite difference method with a hybrid variable-order operator and generalized fourth order Runge-Kutta method are used to solve the optimality system. Numerical examples and comparative studies for testing the applicability of the utilized methods and to show the simplicity of these approximation approaches are presented. Moreover, by using the proposed methods we can concluded that, the model given in this paper describes well the confirmed real data given by WHO about Egypt.     

Surfactant-DNA gel particles: formation and release characteristics

Aqueous mixtures of oppositely charged polyelectrolytes undergo associative phase separation, resulting in coacervation, gelation, or precipitation. This phenomenon has been exploited here to form DNA gel particles by interfacial diffusion. We report on the formation of DNA gel particles by mixing solutions of DNA (either single-stranded (ssDNA) or double-stranded (dsDNA)) with solutions of cationic surfactant cetyltrimetrylammonium bromide (CTAB). By using CTAB, the formation of DNA reservoir gel particles, without adding any kind of cross-linker or organic solvent, has been demonstrated. Particles have been characterized with respect to the degree of DNA entrapment, surface morphology, and secondary structure of DNA in the particles. The swelling/deswelling behavior and the DNA release have been investigated in response to salt additions. Analysis of the data has suggested a higher degree of interaction between ssDNA and the cationic surfactant, confirming the stronger amphiphilic character of the denatured DNA. Fluorescence microscopy studies have suggested that the formation of these particles is associated with a conservation of the secondary structure of DNA.     

Analysis of mutant frequency curves and survival curves applied to the AL hybrid cell system

A model is presented for the statistical analysis of survival curves and mutant frequency curves for a hybrid cell system. The derivation of the model is given in the Appendix, and depends on simple assumptions about the distribution of insults, their repair, and the loss of a marker that is not rescued. A single formula (5) is found which relates a survival curve to the mutant frequency curve, i. e., the response curve for production of mutants per 10(5) survivors induced by a mutagen. The analysis is applied to loss of the a1 gene in AL-J1 hybrid cells submitted to Cesium gamma-rays. Previous experimental data using X-rays was reported by Waldren et al. (1986: Proc. Natl. Acad. Sci, USA 83, 4839.) Also, a derived formula (10), which predicts the probability that in a surviving cell a marker is lost and not rescued, will form the basis for testing the validity of the model in the future using new experimental data.     

Determination of molecular parameters by fitting sedimentation data to finite-element solutions of the Lamm equation.

A method for fitting experimental sedimentation velocity data to finite-element solutions of various models based on the Lamm equation is presented. The method provides initial parameter estimates and guides the user in choosing an appropriate model for the analysis by preprocessing the data with the G(s) method by van Holde and Weischet. For a mixture of multiple solutes in a sample, the method returns the concentrations, the sedimentation (s) and diffusion coefficients (D), and thus the molecular weights (MW) for all solutes, provided the partial specific volumes (v) are known. For nonideal samples displaying concentration-dependent solution behavior, concentration dependency parameters for s(sigma) and D(delta) can be determined. The finite-element solution of the Lamm equation used for this study provides a numerical solution to the differential equation, and does not require empirically adjusted correction terms or any assumptions such as infinitely long cells. Consequently, experimental data from samples that neither clear the meniscus nor exhibit clearly defined plateau absorbances, as well as data from approach-to-equilibrium experiments, can be analyzed with this method with enhanced accuracy when compared to other available methods. The nonlinear least-squares fitting process was accomplished by the use of an adapted version of the "Doesn't Use Derivatives" nonlinear least-squares fitting routine. The effectiveness of the approach is illustrated with experimental data obtained from protein and DNA samples. Where applicable, results are compared to methods utilizing analytical solutions of approximated Lamm equations.