Restriction Map Of DNA Research Articles

An Algorithm for Gene Fragment Reconstruction.

Gene sequencing technology has been playing an important role in many aspects, such as life science, disease medicine and health medicine, particularly in the extremely tough process of fighting against 2019-novel coronavirus. Drawing DNA restriction map is a particularly important technology in genetic biology. The simplified partial digestion method (SPDP), a biological method, has been widely used to cut DNA molecules into DNA fragments and obtain the biological information of each fragment. In this work, we propose an algorithm based on 0–1 planning for the location of restriction sites on a DNA molecule, which is able to solve the problem of DNA fragment reconstruction just based on data of fragments’ length. Two specific examples are presented in detail. Furthermore, based on 1000 groups of original DNA sequences randomly generated, we define the coincidence rate and unique coincidence rate between the reconstructed DNA sequence and the original DNA sequence, and then analyze separately the effect of the number of fragments and the maximum length of DNA fragments on the coincidence rate and unique coincidence rate as defined. The effectiveness of the algorithm is proved. Besides, based on the existing optimization solution obtained, we simulate and discuss the influence of the error by computation method. It turns out that the error of position of one restriction site does not affect other restriction sites and errors of most restriction sites may lead to the failure of sequence reconstruction. Matlab 7.1 program is used to solve feasible solutions of the location of restriction sites, derive DNA fragment sequence and carry out the statistical analysis and error analysis. This paper focuses on basic computer algorithm implementation of rearrangement and sequencing rather than biochemical technology. The innovative application of the mathematical idea of 0–1 planning to DNA sequence mapping construction, to a certain extent, greatly simplifies the difficulty and complexity of calculation and accelerates the process of 'jigsaw' of DNA fragments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12539-021-00419-6.

Correlation of Antibiotic Resistance and Restriction Mapping of Plasmid DNA Isolated from E. coli Causing Urinary Tract Infection

Correlation of Antibiotic Resistance and Restriction Mapping of Plasmid DNA Isolated from E. coli Causing Urinary Tract Infection

DNA hydroxymethylation rate in the AChE and HoxC4 promoter associated with human sperm quality.

The relationship of altered DNA 5'-hydroxymethylation in human spermatozoa with seminal parameters remains unclear. The aim of the study was to investigate the association between the 5'-hydroxymethylcytosine (5hmC) rate in the promoters of acetylcholinesterase (AChE) and homeobox C4 (HoxC4) genes and human sperm concentration/motility. The study population consisted of three groups: asthenozoospermia (AZ), oligoasthenozoospermia (OAZ) and normozoospermia (NZ). The 5hmC rate in the promoter was measured by CCGG loci-dependent MspI/HpaII restriction mapping of glycosylation-modified sperm DNA combined with a hydroxymethylation-specific real-time polymerase chain reaction assay. The 5hmC rate in the AChE promoter in group AZ and OAZ was higher than that in group NZ (p<.05). A weak inverse correlation between 5hmC rate of AChE and sperm motility was observed in all subjects (r=-.172, p<.05). The 5hmC rate in the HoxC4 promoter in group OAZ was lower than that in group NZ (p<.05). These results indicated that altered 5hmC rates of AChE and HoxC4 promoters are associated with low sperm motility and sperm concentration respectively.

Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.

The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl β-D-thiogalactopyranoside (IPTG). Over the course of the next three weeks, students use a battery of approaches including plasmid DNA isolation experiments, restriction maps, and PCR to differentiate between mutation and transformation. The students ultimately come to the conclusion that the changes in phenotypes are due to genetic transformation and not mutation based on the evidence generated over the four-week period. Pre-laboratory tests and post-laboratory tests indicate that this set of exercises is successful in helping students differentiate between transformation and mutation. The laboratory is designed for underclassmen and is a good prerequisite for an apprentice-based research opportunity, although it is not designed as a class based research experience. Potential modifications and future directions of the laboratory based upon student experiences and assessment are presented.

Epidermal Hyperplasia and Appendage Abnormalities in Mice Lacking CD109

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

A new source of cytoplasmic male sterility found in wild beet and its relationship to other CMS types

We found a number of male-sterile plants in a wild beet (Beta vulgaris L. subsp. maritima) accession line, FR4-31. The inheritance study of the male sterility indicated the trait to be of the cytoplasmic type. The mitochondrial genome of FR4-31 proved to lack the male-sterility-associated genes preSatp6 and orf129, which are characteristic of the Owen CMS and I-12CMS(3) cytoplasms of beets, respectively. Instead, the truncated cox2 gene involved in G CMS originating from wild beets was present in the FR4-31 mitochondrial genome. In Southern hybridization using four mitochondrial gene probes, the FR4-31 cytoplasm showed patterns similar to those typical of the G cytoplasm. It is thus likely that the FR4-31 cytoplasm has a different CMS mechanism from both Owen CMS and I-12CMS(3), and that the FR4-31 and G cytoplasms resemble each other closely. A restriction map of the FR4-31 mitochondrial DNA was generated and aligned with those published for the Owen and normal fertile cytoplasms. The FR4-31 mitochondrial genome was revealed to differ extensively in arrangement from the Owen and normal genomes, and the male-sterile Owen and FR4-31 genomes seem to be derived independently from an ancestral genome.

WebFARM: web server for finite automated restriction mapping

Restriction endonucleases are indispensable tools in molecular biology and biotechnology. Type II restriction endonucleases are part of restriction modification systems. DNA fragment extraction and restriction mapping are the basis for several biotechnological activities. WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences. WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases. It will also provide frequency of restriction for each restriction endonuclease. http://webfarm.bioinfoindia.org/

Abstracts of papers presented at the Workshop on Molecular Genetics of the Mouse II August 11-16, 1980 Sandbjerg Slot, Sønderborg, Denmark

The primary purpose of this workshop was to present and discuss recent developments in molecular genetics of mammals, particularly the mouse. The workshop concerned the organization and interaction of genetic information regulating the expression of genes. The purpose of the meeting was also to bring together scientists with knowledge about genetic regulation of gene expression and about restriction mapping and sequencing of genomic DNA to encourage the synthesis of the two rapidly growing fields into specific studies.

Equine herpesvirus 1: characterisation of the first strain isolated in Colombia

This paper describes the isolation and characterisation of equine herpesvirus 1 (EHV-1) in Colombia. The virus was isolated from a nasal swab and an aborted foetus of a pregnant mare imported from Argentina, with clinical signs of rhinopneumonitis. The new strain was characterised through culture and morphological, serological and immunocytochemical studies. Polymerase chain reaction and DNA restriction maps revealed an EHV-1 1P genome. This is the first report on the isolation and characterisation of EHV-1 in Colombia.

RFLP analysis of Hordeum species relationships1

Detailed ribosomal DNA restriction maps were prepared for eight representative plants from Hordeum L. Section Hordeum (two H. bulbosum, H. glaucum, a diploid H. leporinum, a tetraploid H. leporinum, H. murinum, H. spontaneum, H. vulgare) in order to explore species relationships. The four restriction enzymes (EcoRI,SacI, BamHI and HindIII) used in our earlier study were supplemented with six additional ones (BglI, ClaI, KpnI, Pvu II, Sst II and XhoI). Southern blots prepared, using the restriction enzymes singly and in combinations, were probed with the wheat ribosomal probe pTA7l, and hybridization patterns analyzed. A comparison of the derived maps with known maps for wheat, pea and pumpkin showed strong conservation of restriction sites, especially in the ribosomal coding region. The maps suggest that duplication of coding region DNA and its insertion into the spacer region as well as amplification of subrepeats are responsible for variation in rDNA repeat length. Phylogenetic analysis indicated the early divergence of ancestral wheat and Hordeum species, followed by the divergence of the progenitors of H. spontaneum/H. vulgare and the remaining Hordeum species and, most recently, between those of H. glaucum and H. murinum.

Solving large double digestion problems for DNA restriction mapping by using branch-and-bound integer linear programming

The double digestion problem for DNA restriction mapping has been proved to be NP-complete and intractable if the numbers of the DNA fragments become large. Several approaches to the problem have been tested and proved to be effective only for small problems. In this paper, we formulate the problem as a mixed-integer linear program (MIP) by following (Waterman, 1995) in a slightly different form. With this formulation and using state-of-the-art integer programming techniques, we can solve randomly generated problems whose search space sizes are many-magnitude larger than previously reported testing sizes.

Some approximation algorithms for the clique partition problem in weighted interval graphs

Interval graphs play important roles in analysis of DNA chains in Benzer [S. Benzer, On the topology of the genetic fine structure, Proceedings of the National Academy of Sciences of the United States of America 45 (1959) 1607–1620], restriction maps of DNA in Waterman and Griggs [M.S. Waterman, J.R. Griggs, Interval graphs and maps of DNA, Bulletin of Mathematical Biology 48 (2) (1986) 189–195] and other related areas. In this paper, we study a new combinatorial optimization problem, named the minimum clique partition problem with constrained bounds, in weighted interval graphs. For a weighted interval graph G and a bound B , partition the weighted intervals of this graph G into the smallest number of cliques, such that each clique, consisting of some intervals whose intersection on a real line is not empty, has its weight not beyond B . We obtain the following results: (1) this problem is NP -hard in a strong sense, and it cannot be approximated within a factor 3 2 − ε in polynomial time for any ε > 0 ; (2) we design three approximation algorithms with different constant factors for this problem; (3) for the version where all intervals have the same weights, we design an optimal algorithm to solve the problem in linear time.

First restriction and genetic mapping of the genomic DNA of urease-positive thermophilic campylobacters (UPTC), and small restriction fragment sequencing

A restriction and genetic map of urease-positive thermophilic campylobacter (UPTC) CF89-12 genome DNA is constructed using a pulsed-field gel electrophoresis procedure after digestion with SalI and SmaI and Southern blot hybridisation. Each of the six gene fragments (flaA, glyA, lysS, recA, sodB and ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrn operon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrn probes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnp gene (734 bp) bearing a ρ-independent transcriptional termination region, a cluster of five tRNA genes including the putative promoter region, a hypothetical Cj0171-like 507-bp sequence containing an internal termination codon, and a part of the rrn operon including the putative promoter region (4700 bp) are identified. The 507 bp sequence carried both putative transcriptional promoter sequences, including a ribosome binding site upstream of the ATG start codon and a characteristic G9 structure, and a possible ρ-independent transcriptional termination region. A hypothetical Cj0170-like 204-bp sequence containing an internal termination codon also occurred, overlapping partly with the Cj0171-like sequence. Based on nucleotide sequence alignment analysis between the UPTC rrn operon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

Cloning and characterization of directly amplified antiviral gene interferon alpha-2b (HulFNalpha-2b) from human leukocytes chromosomal DNA.

Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation. The interferon alpha gene from human blood samples was amplified, cloned and expressed in E. coli (BL21). Leukocyte chromosomal DNA was used as a source of template DNA. Using specific primers, the gene for HulFNalpha-2b was amplified and inserted into the E. coli vector, pET21b, by ligation of the Hindlll and BamHI linkers of the vector and insert. The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E. coli strain. The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins.

Genome mapping and gene analysis of Antheraea pernyi nucleopolyhedrovirus for improvement of baculovirus expression vector system

We have constructed a genome DNA map of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) and used it to identify target genes for deletion in order to improve the newly developed baculovirus expression vector system. Initially, 50 independent PstI fragments of viral DNA were obtained by shotgun cloning, and both termini of each cloned fragment were sequenced. Then, the sequence data were used for homology search against both nucleotide and amino acid sequences of other NPVs in databases. This homology search allowed us to construct a nearly complete restriction map of a viral DNA with several assumed gaps. Four additional PstI fragments covering the gaps were obtained by PCR amplification, and a complete map of a circular viral DNA, which consisted of 54 PstI fragments, was constructed. The map indicated that the AnpeNPV genome is approximately 130.2 kbp in size and possesses high similarity to the Orgyia pseudotsugata multicapsid NPV (OpMNPV) genome in both sequence and arrangement of genes. Utilizing the genome-wide high similarity between AnpeNPV and OpMNPV, we identified two target genes on the map, namely, cathepsin and chitinase genes, whose products have been proved to be involved in the degradation of recombinant proteins and the liquefaction of virus-infected insect tissues. Comparative sequence analysis of the map also revealed the lack of certain OpMNPV open reading frame (ORF) homologs and the presence of ORFs, whose homologs do not exist in OpMNPV but in other group I NPVs, providing an insight into the position of AnpeNPV in the baculovirus phylogeny.

Genome Mapping and Gene Analysis of Antheraea pernyi Nucleopolyhedrovirus for Improvement of Baculovirus Expression Vector System

We have constructed a genome DNA map of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) and used it to identify target genes for deletion in order to improve the newly developed baculovirus expression vector system. Initially, 50 independent PstI fragments of viral DNA were obtained by shotgun cloning, and both termini of each cloned fragment were sequenced. Then, the sequence data were used for homology search against both nucleotide and amino acid sequences of other NPVs in databases. This homology search allowed us to construct a nearly complete restriction map of a viral DNA with several assumed gaps. Four additional PstI fragments covering the gaps were obtained by PCR amplification, and a complete map of a circular viral DNA, which consisted of 54 PstI fragments, was constructed. The map indicated that the AnpeNPV genome is approximately 130.2 kbp in size and possesses high similarity to the Orgyia pseudotsugata multicapsid NPV (OpMNPV) genome in both sequence and arrangement of genes. Utilizing the genome-wide high similarity between AnpeNPV and OpMNPV, we identified two target genes on the map, namely, cathepsin and chitinase genes, whose products have been proved to be involved in the degradation of recombinant proteins and the liquefaction of virus-infected insect tissues. Comparative sequence analysis of the map also revealed the lack of certain OpMNPV open reading frame (ORF) homologs and the presence of ORFs, whose homologs do not exist in OpMNPV but in other group I NPVs, providing an insight into the position of AnpeNPV in the baculovirus phylogeny.

Mitochondrial DNA restriction map for the Caribbean fruit fly, Anastrepha suspensa, and occurrence of mitochondrial DNA diversity within highly inbred colonies.

A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIlI site. After cloning of the approximately 5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruit flies.

Pulsed‐Field Gel Electrophoresis for Long‐Range Restriction Mapping

This unit describes procedures for generating long-range restriction maps of genomic DNA and for analysis of large insert clones. The basic protocol details restriction digestion of agarose-embedded DNA, PFGE separation, Southern transfer, and hybridization. Support protocols describe the preparation of high-molecular-weight genomic DNA samples in agarose blocks and in agarose microbeads, respectively. Additional support protocols describe the preparation of DNA size standards from l phage and two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. An alternative method of preparing S. cerevisiae size standards using lithium dodecyl sulfate (LiDS) solubilization is provided. The final protocol details the preparation of BAC DNA suitable for digestion, mapping, and sequencing.

Local matching of random restriction maps

Optical mapping is a new technique to generate restriction maps of DNA easily and quickly. DNA restriction maps can be aligned by comparing corresponding restriction fragment lengths. To relate, organize, and analyse these maps it is necessary to rapidly compare maps. The issue of the statistical significance of approximately matching maps then becomes central, as in BLAST with sequence scoring. In this paper, we study the approximation to the distribution of counts of matched regions of specified length when comparing two DNA restriction maps. Distributional results are given to enable us to computep-values and hence to determine whether or not the two restriction maps are related. The key tool used is the Chen-Stein method of Poisson approximation. Certain open problems are described.

Local matching of random restriction maps

Optical mapping is a new technique to generate restriction maps of DNA easily and quickly. DNA restriction maps can be aligned by comparing corresponding restriction fragment lengths. To relate, organize, and analyse these maps it is necessary to rapidly compare maps. The issue of the statistical significance of approximately matching maps then becomes central, as in BLAST with sequence scoring. In this paper, we study the approximation to the distribution of counts of matched regions of specified length when comparing two DNA restriction maps. Distributional results are given to enable us to computep-values and hence to determine whether or not the two restriction maps are related. The key tool used is the Chen-Stein method of Poisson approximation. Certain open problems are described.