Loss Of Restriction Site Research Articles

Multiplex PCR-based RFLP assay for early identification of prevalent Mycobacterium leprae genotypes

Mycobacterium leprae is classified into four SNP genotypes and 16 subtypes (from 1A to 4P) that exhibit phylogeographical association reported from around the world. Among them, genotypes 1D and 3I represent more than 60% of M. leprae strains. Here, we report a new method for M. leprae genotyping which identifies the genotypes 1D and 3I by combining multiplex PCR amplification and restriction fragment length polymorphism (RFLP) of a M. leprae DNA amplicons using AgeI restriction enzyme. Agarose gel electrophoresis showed a deletion of 11 bp only among 3I genotypes by electrophoresis. When this multiplex PCR reaction is subjected to AgeI digestion, successful restriction digestion shows three bands for all the genotypes except 1D where only two bands were observed due to loss of restriction site. This method gives us the advantage of 1-step identification of the two most prevalent strains of M. leprae without using specialized equipments such as the Sanger sequencing system or quantitative PCR.

Genome editing in potato plants by agrobacterium-mediated transient expression of transcription activator-like effector nucleases

Genome editing (also known as targeted mutation) has promise for molecular breeding. Compared with the CRISPR/Cas9 system, the transcription activator-like effector nucleases (TALENs) have likely a lesser off-target rate in genome editing. Both a rapid test system for the functionality of designed TALENs and an effective delivery system for introducing the TALENs to plants are critical for successful target mutation. TALENs have usually been tested in protoplasts or introduced to plants with viral vectors. However, plant regeneration from protoplast culture can generate extensive somatic variation. Viral vectors are not always available, and plants edited by these vectors usually require virus elimination. Here, we used a non-viral, Agrobacterium-mediated transient expression approach, to serve both rapid test and effective delivery of TALENs into two vegetatively propagated potato cultivars, Solanum tuberosum Russet Burbank and Shepody. Two TALENs with different molecular weights (22 and 27 aa-repeat modules) were expressed to target two endogenous genes (starch branching enzyme and an acid invertase) by Agrobacterium-mediated infiltration (agroinfiltration) into leaves of potato plants. The infiltrated leaf DNA was analyzed using restriction site loss assay and subsequent DNA sequencing. Deep sequencing of these tetraploid cultivars was also conducted to determine the zygosity at the targeted chromosomal loci. TALENs, with different molecular weights, successfully agroinfiltrated and induced mutations at both targeted loci.

Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions.

Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.

CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is a highly accessible genome editing tool. Here we demonstrate its potential for use in Taraxacum species. Taraxacum kok-saghyz (TK, Rubber Dandelion) is notable for its ability to produce high molecular weight rubber in its roots and promise as an alternative source of natural rubber. In order to accelerate the domestication of TK, we have established a simple strategy to deploy CRISPR/Cas9 in this species. A critical gene encoding fructan:fructan 1-fructosyltransferase (1-FFT), implicated in inulin biosynthesis was selected as the target, as inulin is an expected antagonist of rubber production. TK plantlets were inoculated with Agrobacterium rhizogenes harboring a plasmid encoding a Cas9 and sgRNA (single guide RNA) targeting TK 1-FFT. We were able to rapidly induce hairy roots harboring knockout alleles without the selection of stable, herbicide or antibiotic resistant transformants. Mutagenesis was affirmed by observing a loss of restriction sites within 1-FFT, followed by sequencing. Of 11 hairy root samples tested, 10 showed the presence of genome editing, with mutation rates as high as 88.9%, suggesting a high efficiency mutagenesis induced by CRISPR/Cas9 via A. rhizogenes-mediated transformation. Whole TK plants were regenerated from hairy roots harboring knockout alleles. The regenerated plants contained knockout alleles, with mutation rates as high as 80.0%. TK plants with edited genomes were obtained within 10 weeks. By omitting a selection step, it was possible to generate edited TK plants lacking stably transformed CRISPR elements, which may potentially reduce off-target mutagenesis. Application of high efficiency CRISPR/Cas9 genome editing will facilitate the domestication and commercialization of TK as a rubber producing crop, and may accelerate basic research on the regulation of rubber biosynthesis.

Mutación en el gen gyrA de aislamientos hospitalarios de Acinetobacter baumannii en Montería, Colombia

ObjectiveDetermine mutations in the gyrA gene associated to resistance to fluoroquinolones in A. baumannii. Materials and methodsFrom August, 2005 to February, 2007, 23 A. baumannii isolates were collected in a third level private clinic in Monteria. Research on gyrA, parC and AdeB genes was carried out, and the latter encodes for the efflux pump. DNA sequencing was performed, and the GenBank database and BLASTX search engine were used for sequence analysis. ResultsAmplification of the gyrA gene in A. baumannii isolates generated a 343pb fragment which presented loss of restriction site with the enzyme HinfI in 12/23 (52.1%) of the isolates resistant to fluoroquinolones. The fragment sequencing showed mutation characterized by the change of Ser-83 to Leu GenBank acces code EU886740. None of the isolates showed mutations in the parC gene or presence of the AdeB efflux pump. ConclusionThe A. baumannii isolates resistant to fluoroquinolones suggest that mutation of the gyrA gene encoding the serine amino acid change to leucine at codon 83 of these isolates is responsible or at least contributes to the mentioned resistance to fluoroquinolones.

Epstein-Barr virus latent membrane protein-1 (LMP-1) 30-bp deletion and Xho I-loss is associated with type III nasopharyngeal carcinoma in Malaysia

BackgroundNasopharyngeal carcinoma (NPC) is a human epithelial tumour with high prevalence amongst Chinese in Southern China and South East Asia and is associated with the Epstein-Barr virus (EBV). The viral genome harbours an oncogene, namely, the latent membrane protein 1 (LMP1) gene and known variants such as the 30-bp deletion and loss of XhoI restriction site have been found. Less is known about the relationship between these variants and the population characteristics and histological type.MethodsIn this study, the EBV LMP1 gene variants from 42 NPC and 10 non-malignant archived formalin fixed, paraffin-embedded tissues, as well as plasma from another 35 patients with nasopharyngeal carcinoma were determined by using Polymerase Chain Reaction (PCR). Statistical analysis was performed by using SPSS programme.ResultsLMP1 30-bp deletion was detected in 19/34 (55.9%) of NPC tissues, 7/29 (24.1%) of plasma but absent in non-malignant tissues (8/8). Coexistence of variants with and without 30bp deletion was found only in 5/29 (17.2%) plasma samples but not in NPC tissues. The loss of XhoI restriction site in LMP1 gene was found in 34/39 (87.2%) of the NPC tissues and 11/30 (36.7%) of plasma samples. None of the non-malignant nasopharyngeal tissues (8/8) harbour XhoI-loss variants. LMP1 30-bp deletion was detected in 16/18 Chinese versus 3/15 Malays and 13/16 type III (undifferentiated carcinoma) versus 1/6 type I (keratinizing squamous cell carcinoma). XhoI-loss was found in 19/19 Chinese versus 14/19 Malays and 18/18 type III (undifferentiated) versus 2/5 type I (keratinizing squamous cell carcinoma). Statistical analysis showed that these variants were associated with ethnic race (30-bp deletion, p < 0.05; XhoI-loss, p = 0.046) and histological type of NPC (30-bp deletion, p = 0.011; XhoI-loss, p = 0.006). Nineteen out of 32 NPC tissues (19/32; 59.4%) and 6/24 (25%) of plasma samples showed the coexistence of both the 30-bp deletion and the loss of XhoI restriction site. A significant relationship was found with the Chinese race but not histological type.ConclusionThe incidence rate of 56% for LMP1 30-bp deletion was lower compared to previously reported rates of 75–100% in NPC tissues. Coexistence of variants with and without 30-bp deletion was found only in 5/29 plasma samples. The incidence rate of XhoI restriction site loss in NPC was comparable to other studies from endemic regions such as Southern China. For the first time, the presence of LMP1 30-bp deletion or XhoI-loss was associated with the Chinese race and type III NPC. Both these variants were not found in non-malignant tissues. The influence of these variants on disease progression and outcome in Chinese and type III NPC requires further investigation.

Loss of restriction site DdeI, used for avian molecular sexing, in Oreophasis derbianus.

Molecular sexing is a rapid and safe procedure for bird sex determination. Two universal methods based on the amplification of a chromo-helicase-DNA-Binding 1 (CHD) gene region, located in both sexual chromosomes (Z and W), have been established. We found that molecular sexing of Oreophasis derbianus failed by using these two procedures. One of them is based on a restriction site located in CHD1W gene but absent in CHD1Z. The DdeI restriction site, used successfully to determine gender in several bird species, was found to be lost because of nucleotide change in O. derbianus. This change created a new restriction site, NlaIII, that was successfully applied for sexing this endangered bird.

A modified rapid method of nucleic acid isolation from suspension of matured virus: applied in restriction analysis of DNA from an adenovirus prototype strain and a patient isolate

This report describes a method for the isolation of nucleic acid from a suspension of matured virus. Nucleic acid (DNA) was isolated from a prototype strain of adenovirus type 7 and a clinical isolate of adenovirus type 7. Instead of the usual method of ultracentifugation, a filtration method was applied to concentrate the virus rapidly and nucleic acid was then isolated by a standard phenol/chloroform/isoamyl-alcohol extraction procedure. The DNA was found to be sufficiently purified to generate a reproducible restriction endonuclease digestion pattern. The clinical isolate of adenovirus type 7 revealed loss of restriction site for the endonuclease HindIII when compared with the prototype strain.

Determination of Bias in the Relative Abundance of Oligonucleotides in DNA Sequences

Different statistical measures of bias of oligonucleotide sequences in DNA sequences were compared, both by theoretical analysis and according to their abilities to predict the relative abundances of oligonucleotides in the genome of Escherichia coli. The expected frequency of an oligonucleotide calculated from a maximal order Markov model was shown to be a degenerate case of the expected frequency calculated from biases of all subwords arising when noncontiguous subwords exhibit no bias. Since (at least in E. coli) noncontiguous sequences exhibit significant bias, the total compositional bias approach is expected to represent biases in genomic sequences more faithfully than Markov approaches. In fact, the efficacy of statistics based on Markov analysis even at the highest order were inferior in predicting actual frequencies of oligonucleotides to methods that factored out biases of internal subwords with gaps. Using total compositional bias as a measure of relative abundance, tetranucleotide and hexanucleotide palindromes were found to be distributed differently from nonpalindromic sequences, with their means shifted somewhat towards underrepresentation. A subpopulation of palindromic hexanucleotides, however, was highly underrepresented, and this group consisted almost entirely of targets for Type II restriction enzymes found within strains of E. coli. Sites recognized by Type I endonucleases from related strains were not markedly biased, and with pentanucleotides, palindromic and nonpalindromic sequences had nearly identical distributions. The loss of restriction sites may be explained by the free transfer of plasmids encoding restriction enzymes and episodic selection for the presence of the enzymes.

ESTUDIO DE UNA PRENDA TEXTIL ASOCIADA AL INCA EN LA COSTA NORTE DE CHILE (CAMARONES 9): LAS "MANTAS" QUE ENVUELVEN LOS CUERPOS

Mitochondrial DNA polymorphisms have been extensively used in the reconstruction of the human evolutionary history owing to its maternal inheritance, lack of recombination, and high mutation rate relative to nuclear DNA. In the Americas, the primary goals of the mitochondrial DNA analysis have been to determine the origins, relationships and migrational patterns of New World populations. In this respect, the study of the frequency distribution of the four founding Amerindian haplogroups defined by Torroni et al. (1992), has proven useful. Each haplogroups can be characterized by a specific mtDNA marker: an Hae III restriction site gain at np. 663 for haplogroup A, the 9 bp. deletion in the COII/tRNAlys region for haplogroup B, a Hinc II restriction site loss at np. 13259 for haplogroup C and an Alu I restriction site loss at np. 5176 for haplogroup D. In order to expand the genetic data related to the peopling of South America, we studied mtDNA polymorphisms in five skeletal remains from the Azapa valley (AZ-71 site) dated 3000 to 2000 years BP, and compared them with the extant population of San Pedro de Atacama in northern Chile. The mtDNA of contemporary individuals was extracted by standard procedures (Lahiri DK et al. 1988). Ancient mtDNA was extracted from bone remains following an adapted protocol from Hoss & Paabo (1993). First, to destroy possible contaminant DNA on the surface of remain, bone fragments were exposed to UV irradiation. The samples were crushed and then powdered in a refrigerated mill. The powdered samples were incubated in 8 ml of 0.5 M EDTA solution for decalcification for 24 hours, and collected by centrifugation. The precipitate was suspended in 5 ml of lysis buffer (5.5 M guanidinium thiocyanate, 50 mM Tris-HCl pH: 6.4, 20 mM EDTA and 1.3 % Triton X100) and incubated at 60oC for several hours, with sporadic shaker. After centrifugation, the supernatant was removed to another tube and mixed with equal volume of lysis buffer. A 40 ml aliquot of silica suspension was added, and the mixture was incubated for 15 minutes at room temperature. After centrifugation the silica pellet was washed successively with washing buffer, 70% ethanol and acetone. Finally the pellet was dried, and the ancient DNA was eluted at 56oC in 100 ml TE buffer. The extant population of San Pedro de Atacama shows the four Amerindian haplotypes, analyzed by Amp-RFLP, with the following distribution: haplogroup A, 8.3%; haplogroup B, 62.5%; haplogroup C, 25% and haplogroup D, 4.2%. This distribution is very similar to the one described previously for Aymara populations from the North of Chile, and is in consistent with findings in other Andean high altitude populations. We identified, furthermore the sequence of the mtDNA hypervariable region I (position 16081 to 16409) for 23 individuals. Results revealed consistent changes described for these haplogroups.

Comparative studies of the structure of chloroplast DNA from four species of Oryza: cloning and physical maps

Chloroplast DNAs (ctDNAs) were prepared from the mature green leaves of three species in the genus Oryza, namely, O. punctata (genome type BB), O. offici-nalis (CC), and O. australiensis (EE). After digestion with restriction enzymes, ctDNAs were cloned into a lambda phage vector and overlapping clone banks of the entire chloroplast genome from each of the three Oryza species were obtained. BamHI and PstI restriction maps of the ctDNAs were constructed, and the structures of the ctDNAs from O. sativa and the other three Oryza species were compared. Two types of variation were noted: the gain or loss of restriction sites, and deletion or insertion of nucleotides. We detected two independent deletions in the BamHI-3/PstI-3 fragment of O. punctata and in the BamHI-5/PstI-11 fragment of O. officinalis, each of which was shorter than the respective fragment from O. sativa, and the deletions were located in spacer regions. Short direct-repeat sequences were detected at the border of both deletions, indicating that these deletions were results of intramolecular recombination mediated by these direct repeats. Further analysis on distribution of those deletions among 15 Oryza species revealed that the deletions found in this study represent genotype-specific variations.

Phylogenetic studies of acridoid grasshoppers comparing 2n2‐ and 4‐base recognizing endonucleases

AbstractThe frequency of loss of restriction sites may be a limiting factor in the application of RFLPA to phylogenetic investigations of higher taxonomic categories. One purpose of this work is to ascertain whether the applicability of RFLPA can be extended by the use of a class of restriction endonucleases which will here be termed 2n2 cutters, as any base may occupy the middle position of the recognition sequence. Such endonucleases should be less susceptible than, say, 4‐base cutters to the redundancy of the genetic code when this position coincides with the third base of a codon. Here the two types of endonucleases are compared by internal statistics summarizing the phylogenetic content of the data and by the concordance of their resulting dendrograms to six known relationships of thirteen acridid and eumustacid grasshoppers. Data were gathered for a mtDNA probe (expected to show redundancy effects) and an rDNA probe (not expected to show such effects). Phenetic and cladistic analyses were performed. There was no evidence that the 2n2‐cutting endonucleases produced more accurate dendrograms. In fact, in contrast with expectations and with, as yet, no apparent explanation, trees produced by 4‐base cutting endonucleases had greater concordance to present best estimates of grasshopper relationships. The second main purpose of the paper was to extend molecular studies of the phylogeny of the Acrididae. The two main results here were the finding of a close relationship between the Oxyinae and the Catantopinae and the apparent absence of a major endemic Australian radiation in the Acridinae.

Distribution of restriction site polymorphism within the chloroplast genome of the genus Glycine, subgenus Soja

Restriction fragment length polymorphisms (RFLPs) have been used to detect intragenic sequence diversity in Glycine subgenus soja chloroplast DNA. The distribution of these RFLPs allow Glycine max and G. soja accessions to be grouped according to cytoplasmic genetic relatedness. DNA clones from mung bean chloroplast DNA were used to locate the RFLPs to specific regions of the chloroplast genome. In the course of the experiments, several previously unobserved RFLPs were also identified. At least six molecular changes were detected, including both restriction site loss or gain and insertion/deletion events. Three of the fragment polymorphisms detected are due to changes in the juncture region between one inverted repeat region and the large single-copy region. Probes detecting polymorphisms in three representative soybean genotypes were used to screen additional cultivars and Plant Introductions. The distribution of RFLP patterns in these accessions were consistent with the patterns of previously described cytoplasmic groupings, with the exception of one accession, which formed a new plastome group.

Distribution and evolution of a tandemly repeated DNA sequence in the family brassicaceae

A tandemly repeated DNA sequence present in several species of the family Brassicaceae has been characterized. Digestion of the genomic DNA from 17 species from this family revealed the presence of tandem repeats of about 175 bp monomeric size in 6 of the 11 investigated genera. All investigated genera within the tribe Brassiceae contained the sequence. TheBrassica napus Hind III 175-bp monomeric repeat unit was isolated and used as a probe in filter hybridizations to DNA from the other species. The sequence was found under highly stringent hybridization conditions in all of the sevenBrassica species analyzed, in the closely related genusEruca, and in the more distant genusCapsella. The generaRaphanus, Sinapis, andCrambe hybridized with the probe under moderately stringent conditions. The degree of hybridization increased with a further drop in hybridization stringency inRaphanus, Sinapis, andCrambe, indicating that sequence divergence has occurred in these genera relative toB. napus. The distribution of the various sequence multimers indicated a random and similar loss of restriction sites in all species hybridizing under high stringency. The copy number per haploid genome in theBrassica species varied between 1.7×105 and 3.0×105, except inB. nigra, where only 3.0×104 copies were found.

EVOLUTIONARY INFERENCES FROM RESTRICTION MAPS OF MITOCHONDRIAL DNA FROM NINE TAXA OF XENOPUS FROGS

Restriction endonuclease cleavage maps were prepared by the double digestion method for mitochondrial DNAs (mtDNAs) purified from Xenopus borealis, X. clivii, X. fraseri, X. muelleri, X. ruwenzoriensis, X. vestitus, X. laevis victorianus, X. l. laevis, and a variant of X. laevis designated X. laevis "davis." An average of 21 cleavage sites per genome were mapped with 11 restriction endonucleases. Among the four invariant sites found are three conserved not only among the Xenopus mtDNAs tested but also among nearly all vertebrate mtDNAs examined to date. Two of these are Sac II sites in the 12S and 16S ribosomal RNA genes, and one is a Hpa I site in the gene for asparagine transfer RNA. These three sites permit the alignment and comparison of mtDNAs from different vertebrate classes. Although most of the differences observed among the Xenopus maps are attributable to point mutations causing gain or loss of restriction sites, the maps also differ by three large length mutations in or near the displacement loop. Phylogenetic analysis of 30 informative sites suggests that those members of the laevis species-group that have 36 chromosomes per somatic cell can be divided into three subgroups: 1) X. borealis, X. clivii, and perhaps X. fraseri (the "borealis" subgroup), 2) X. muelleri, and 3) the subspecies of X, laevis. The mtDNA of the hexaploid (2n = 108) species, X. ruwenzoriensis, is most similar to that of taxa in the latter two subgroups, which contrasts with the morphological similarity of this species to X. fraseri. X. ruwenzoriensis may be an allopolyploid with a mother (the contributor of the cytoplasmic mtDNA genome) on the X. laevis or X. muelleri lineage and a father on the X. fraseri lineage. We present a model showing how mtDNA and nuclear genomes can yield contrasting phytogenies for species-groups that have undergone several rounds of interspecific hybridization. Comparison of mitochondrial and nuclear sequence divergences suggests that Xenopus mtDNA, like that of mammals and birds, evolves faster than nuclear DNA. Genetic distances among mtDNAs of Xenopus species are very large, generally approaching or exceeding one substitution per nucleotide.

Southern analysis of genomic alterations in gamma-ray-induced aprt- hamster cell mutants.

The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus.

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences.