EcoRI Cleavage Sites Research Articles

A combined approach of rolling-circle amplification-single site restriction endonuclease digestion followed by next generation sequencing to characterize the whole genome and intra-host variants of human Torque teno virus

Torque Teno Virus (TTV) was initially associated with post-transfusion hepatitis, but growing evidence of its ubiquity in humans is compatible to no apparent clinical significance. TTV is a small non-enveloped virus with a circular single-negative-stranded DNA genome, belonging to the Anelloviridae family. Currently, TTVs are divided in seven phylogenetic groups and are further classified into 21 species. Studies about diversity of TTV in different conditions are receiving increasing interest and in this sense, sequencing of whole genomes for better genetic characterization becomes even more important. Since its discovery in 1997, few TTV complete genomes have been reported worldwide. This is probably due, among other reasons, to the great genetic heterogeneity among TTV strains that prevents its amplification and sequencing by conventional PCR and cloning methods. In addition, although metagenomics approach is useful in these cases, it remains a challenging tool for viromic analysis. With the aim of contributing to the expansion of the TTV whole genomes dataset and to study intra-host variants, we employed a methodology that combined a rolling-circle amplification approach followed by EcoRI digestion, generating a DNA fragment of ∼4Kb consistent with TTV genome length which was sequenced by Illumina next generation sequencing. A genogroup 3 full-length consensus TTV genome was obtained and co-infection with other species (at least those with a single EcoRI cleavage site) was not identified. Additionally, bioinformatics analysis allowed to identify the spectrum of TTV intra-host variants which provides evidence of a complex evolution dynamics of these DNA circular viruses, similarly to what occurs with RNA viruses.

Identification of Regulatory-RNAs for Alternative Splicing of Ron Proto-Oncogene.

RON receptor tyrosine kinase is a proto-oncogene that induces cell migration and matrix invasion. RONΔ160 protein, which is produced by exclusion of exon 5 and 6, promotes cell migration, matrix invasion and protection from apoptosis. Alternative splicing regulation of exon 5 and 6 is not well understood. In this manuscript, we identified several new RNA regulatory elements for alternative splicing of Ron proto-oncogene. Firstly, we demonstrated that RNA sequences from EcoRI cleavage sites regulate alternative splicing of Ron exon 5 and 6. Secondly, we showed that the ~30 nt RNA at upstream end of exon 4 and the ~33 nt RNA at downstream end of exon 7 also modulate splicing of exon 5 and 6. Thirdly, our results indicate that the RNA sequences of the ends in exon 4 and 7 are required for the regulatory functions of the RNA from restriction enzyme cleavage sites. Our results provide a new insight for regulation of alternative splicing of Ron proto-oncogene.

Cell Cycle Regulation of Microtubule Interactomes: Multi-layered Regulation Is Critical for the Interphase/Mitosis Transition

Microtubules dramatically change their dynamics and organization at the entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we used an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that the suppression of the microtubule affinity of this mitotic MAP in combination with nuclear localization is essential for microtubule organization in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.

A Pseudo-Full Mutation Identified in Fragile X Assay Reveals a Novel Base Change Abolishing an EcoRI Restriction Site

Diagnostic testing for the fragile X syndrome is designed to detect the most common mutation, a CGG expansion in the 5'-untranslated region of the fragile X mental retardation (FMRI) gene. PCR can determine the number of CGG repeats less than 100, whereas Southern analysis can detect large premutations, full mutations, and their methylation status. Bands larger than 5.8 kb observed via Southern analysis are usually considered a methylated full mutation, causing fragile X syndrome in males and varied clinical presentations in females. We observed a 10.9-kb band on a Southern blot assay from an autistic girl with language delay. Further investigation identified a novel G-to-A transition at an EcoRI cleavage site, upstream of the CGG repeat region of the FMRI gene. This base change abolished the EcoRI restriction site, resulting in a 10.9-kb pseudo-full mutation. This G-to-A base change has not been previously reported and was not identified in a subsequent analysis of 105 male and 30 female patient samples. The clear 10.9-kb band detected on a Southern blot assay for fragile X syndrome mimics a large, methylated full mutation, which could result in a misdiagnosis without the benefit of family studies and further testing.

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155.

In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155.

Detection of Chlamydia trachomatis by the polymerase chain reaction in the cervices of women with acute salpingitis

OBJECTIVE: Our objective was to determine whether an increased prevalence of Chlamydia trachomatis could be detected by the polymerase chain reaction as opposed to culture in the cervices of women with acute salpingitis. STUDY DESIGN: Endocervical samples from 15 women with laparoscopy-verified acute salpingitis and 20 women seeking medical help for conditions other than pelvic pain were tested for Chlamydia trachomatis with the polymerase chain reaction. The oligonucleotide primer pairs used were specific for a 144 bp region of the major outer membrane protein that contained a single Eco RI endonuclease cleavage site. The detection of a 144 bp band that was cleaved by Eco RI to a 103 bp band denoted a Chlamydia trachomatis -positive sample. Cervical samples were cultured for Chlamydia trachomatis with the use of McCoy cells. The lymphocyte proliferative response to Chlamydia trachomatis elementary bodies was also determined. RESULTS: Nine of the 15 women (60%) with salpingitis had positive results when tested with the polymerase chain reaction for cervical Chlamydia trachomatis . Only two of these women (13%), both of whom had positive results when tested with the polymerase chain reaction, had cultures that were positive for Chlamydia trachomatis ( p Chlamydia . Those women plus two women with unexplained recurrent abortions had positive polymerase chain reaction test results for Chlamydia trachomatis . A lymphocyte proliferative response to Chlamydia trachomatis was detected in five of eight women with salpingitis, as well as three of the other four patients, all of whom had positive polymerase chain reaction test results; lymphocytes from the remaining women were unresponsive. Follow-up cervical samples were obtained 4 to 6 months after treatment from six of the patients with salpingitis who had positive polymerase chain reaction test results; at that time five had negative polymerase chain reaction test results for Chlamydia trachomatis . CONCLUSION: The polymerase chain reaction appeared to be more sensitive and more rapid than culture in detecting Chlamydia trachomatis in the cervices of women with acute salpingitis. This assay may be of value for the early diagnosis of chlamydial infections.

A novel intergenic site for integration and expression of foreign genes in the genome of Pseudorabies virus

Restriction enzyme analysis of DNA of a number of Pseudorabies virus (PRV) single plaque isolates revealed in several cases the existence of a unique EcoRI cleavage site, which has not been observed in PRV DNA before. This EcoRI site was mapped to the right end of the unique long region of the PRV genome, in BamHI-fragment 6. Sequence analysis of this region demonstrated the presence of an 11 bp tandem repeat in variable copy numbers in different PRV strains, suggesting the creation of the EcoRI recognition site by a recombinational event. The occurrence of variable reiterations and Northern blot analysis indicated an intergenic region. We therefore, used this site for integration and expression of heterologous DNA (the multiple cloning site of phage M13 and the E. coli lacZ gene). Viable PRV recombinants could be obtained which showed no detectable differences in virus growth in vitro compared to wild-type PRV. The novel insertion site can be used for the construction of PRV recombinants expressing foreign genes without apparent impairment of PRV genes.

Apolipoprotein B gene DNA polymorphisms are associated with macro‐ and microangiopathy in non‐insulin‐dependent diabetes mellitus

The relationship between diabetic macroangiopathy or microangiopathy and apolipoprotein B (apoB) polymorphism was studied in 139 male and 129 female patients with non-insulin-dependent diabetes (NIDDM) mellitus, comprising consecutive patients with poor diabetic control (HBA1 13.2% +/- 2.7 (SD)) referred to our hospital. Plasma cholesterol and triglyceride concentrations were higher in the patients who were homozygous for the X2 allele (presence of XbaI cleavage site). Patients with the X1 allele (absence of XbaI cleavage site) tended to have a higher frequency of macroangiopathy, although the differences were not statistically significant. There was no difference in the prevalence of microangiopathy between the groups. In subjects with only an R1 allele (= R+; homozygous for the presence of EcoRI cleavage site) the prevalence of coronary heart disease (CHD) was observed to be high (61.9%) as compared to the subjects possessing an R2 allele (= R-; homozygous or heterozygous for the absence of the EcoRI cleavage site) (46.7%; p < 0.02). When the polymorphisms XbaI (subjects homozygous for the absence of the cutting site = X+; subjects homozygous or heterozygous for the presence of the cutting site = X-) and EcoRI were combined, the prevalence of macroangiopathy was observed to be high in X+R+ (80.0%) as compared with X+R- (44.2%), X-R+ (56.8%) and X-R- (50.0%) (p < 0.03). The prevalence of macroangiopathy tended to be particularly high in patients with the apoprotein E4 allele (phenotype E4/4 or E4/3), combined with either X+ or R+.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular analysis of abnormal satellite I DNA from a BUF/Mna rat thymoma.

BUF/Mna rats develop spontaneous thymomas in an autosomal dominant manner. We constructed recombinant plasmid library of 90 and 185 base-paired (bp) satellite I DNA fragments isolated from BUF/Mna rat thymoma DNA. Four unusual clones containing 93, 95, 95, and 173 bp inserts were isolated by colony hybridization with Wistar rat satellite I DNA. Nucleotide sequence analysis of the inserts of the 4 clones revealed abnormal sequence organization and unusual subunit structure of the rat satellite I DNA. Sequence comparisons between normal and abnormal satellite I DNA suggested that the unusual subunit structure could be generated by the change of the Hinf I recognition sequence to an Eco RI cleavage site, in addition to random deletions, insertions and base substitutions. The heptanucleotide sequence TGGGAAC, which is strictly conserved in normal subunits, was completely lost in all these clones. Southern blot hybridization revealed the amplification of abnormal satellite I DNA in BUF/Mna rat thymomas.

Variation in the restriction fragments of 18S–26S rRNA loci in South American Elymus (Triticeae)

The repeat units of the 18S–5.8S–26S ribosomal genes (rDNA) of 15 South American and 6 Asiatic and North American species of Elymus were shown to vary between 8.7 and 11.3 kb among species. Interspecific variation in the EcoRI and BamHI cleavage sites was observed. BamHI restriction sites were present in the subrepeats in the intergenic spacers in rDNA from diploid Hordeum (H genome) and absent in the diploid Pseudoroegneria (S genome) and in nearly all the tetraploid species of Elymus (SH genomes). Tetraploid Elymus species have only two pairs of chromosomes with secondary constrictions similar to those found in Pseudoroegneria and a maximum of two different repeat unit length classes. These results suggest that the rDNA in the H genome was lost during the evolution of the tetraploid Elymus. However, the additional H genome present in the hexaploid Elymus with genome formula SSHHH′H′ still show active ribosomal genes. Nucleolar organizer activity was also observed in rDNA from both the S and H genomes in artificial SS × HH amphiploids, suggesting no complete nucleolar dominance (amphiplasty) between the S and H genomes.Key words: Elymus, ribosomal genes, RFLP, nucleolus.

The maternally inherited regulation of P elements in Drosophila melanogaster can be elicited by two P copies at cytological site 1A on the X chromosome.

Two P elements, inserted at the cytological site 1A on an X chromosome from an Drosophila melanogaster natural population (Lerik, USSR), were isolated by genetic methods to determine if they are sufficient to cause the P cytotype, the cellular condition that regulates the P family of transposable element. The resulting "Lerik P(1A)" line (abbreviated "Lk-P(1A)") carries only one P element in situ hybridization site but genomic Southern analysis indicates that this site contains two, probably full length, P copies separated by at least one EcoRI cleavage site. Because the Lk-P(1A) line shows some transposase activity, at least one of these two P elements is autonomous. The Lk-P(1A) line fully represses germline P element activity as judged by the GD sterility and snw hypermutability assays; this result shows that the P cytotype can be elicited by only two P element copies. However, the Lk-P(1A) line does not fully repress delta 2-3(99B) transposase activity in the soma, although it fully represses delta 2-3(99B) transposase activity in the germline (delta 2-3(99B) is an in vitro modified P element that produces a high level of transposase activity in both the germline and the soma). The germline regulatory properties of the Lk-P(1A) line are maternally transmitted, even when the delta 2-3(99B) element is used as the source of transposase. By contrast, the partial regulation of delta 2-3(99B) somatic activity is chromosomally inherited. These results suggest that the regulatory P elements of the Lk-P(1A) line are inserted near a germline-specific enhancer.

Polymerase Chain Reaction (PCR) amplification for the detection of porcine parvovirus

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species

Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1-3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A - H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B - H. leporinum; Map C - H. vulgare, H. spontaneum, H. agriocrithon; Map D - H. chilense, H. bogdanii; and Map E - remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.

Two new genome types of adenovirus 7c.

Adenovirus type 7 is the type most frequently associated with serious disease. Eighteen different genome types of adenovirus type 7 had been reported up to October 1986. The genome type Ad7c, based on the restriction enzyme profiles of SmaI and BamHI, has been reported from Europe prior to 1969 and more recently from South Africa. Here, we report two new genome types of adenovirus 7 c that have not previously been identified and that have been isolated in South Africa between 1975 and 1986 from children with postmeasles pneumonia. The two new genome types differ from the prototype Ad7c virus in having two (Ad7c1) or one (Ad7c2) extra cleavage sites for the restriction endonuclease EcoRI. These sites have been located at 3.68kb and 5.32kb from the left terminus of the genome map published for the prototype Ad7c strain. A strain resembling the prototype Ad7c was also isolated in 1986 from a case of post measles pneumonia.

The structure and cloning of orf virus DNA

A map of cleavage sites for the restriction endonucleases EcoRI, HindIII, BamHI, HpaI, and KpnI for a New Zealand strain of orf virus (NZ2) DNA has been deduced. Also, the entire genome, apart from approximately 0.1 kbp at each end, has been cloned into various vectors. The genome is 139 kbp in length and, in common with other poxviruses, has inverted terminal repetitions and crosslinked ends.

Cloning, physical mapping and cross-hybridization of the canine adenovirus types 1 and 2 genomes

Cloned HindIII fragments of the type 1 and type 2 canine adenovirus (canAd) genomes were mapped with respect to the BamHI, EcoRI, HincII, PaeR7 and PstI restriction endonuclease cleavage sites. Considering the large differences found between the canAd-1 and canAd-2 DNA molecules, in terms of number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the canAd-1 and canAd-2 physical maps. Both viruses are genetically related to the extent of 57%. Our results confirm the existence of two distinct Ad species in the dog.

Solution structure of d-GAATTCGAATTC by 2D NMR: A new approach to determination of sugar geometries in DNA segments

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d-GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare Ol′-endo geometry. The EcoRI cleavage sites (between G and A) in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2′-endo and C4′-endo.

IS222, a new insertion element associated with the genome of Pseudomonas aeruginosa.

A new insertion element, IS222, was identified to be associated with the DNA of a mutant strain of the converting Pseudomonas aeruginosa bacteriophage D3. The insertion sequence was 1,350 base pairs in size and possessed terminal inverted repeats. The nucleotide sequence contained single cleavage sites for EcoRI and PvuI but none for BamHI, PstI, HindIII, SmaI, or SalI. By Southern hybridization analysis, no homology was found with genomic DNA from P. aeruginosa PAT or Escherichia coli. Genomic DNA from the phage host, P. aeruginosa PAO, contained two sequences homologous to IS222.

Resistance to mercury and to cadmium in chromosomally resistant Staphylococcus aureus.

Apparently chromosomally located mercury resistance determinants in five methicillin-resistant Staphylococcus aureus strains of different geographical origin were structurally homologous to plasmid-located mercury resistance determinants in S. aureus. These were all located on a 6.3-kilobase (kb) Bg/II fragment, as evident from Southern hybridization experiments with the 6.3-kb Bg/II fragment of plasmid pI258 as the probe. These methicillin-resistant S. aureus strains exhibited similar phage susceptibility patterns and biochemical reactions. They differed, however, in the DNA location of the mercury resistance determinants, as evidenced by neighboring cleavage sites for restriction endonucleases EcoRI, HindIII, and PstI. In an environmental (nonhospital) strain in which mercury resistance was also apparently chromosomally conferred, these determinants were also homologous to pI258 DNA, but they were located on a 6.6-kb Bg/II fragment. Cadmium resistance determinants in the five methicillin-resistant S. aureus strains and the environmental S. aureus strain were not similar to the known plasmid-located determinants cadA and cadB. Cd2+ resistance was based on an efflux mechanism for Cd2+. However, no parallel resistance to zinc was conferred. The 3.2-kb XbaI-Bg/II fragment obtained from plasmid pI258 and used as a cadA-specific probe did not hybridize to total DNA digests of the strains with apparently chromosomally determined cadmium resistance.

Highly mobile DNA segment of IncI alpha plasmid R64: a clustered inversion region.

When R64 DNA was digested with EcoRI, two DNA fragments not equimolar to the plasmid DNA were produced. A DNA region including these fragments was cloned (pKK009), and the pKK009 DNA sample was found to be a mixture of six or more DNA species with EcoRI, PstI, and AvaI cleavage sites at different positions, suggesting a complex rearrangement of DNA. When a part of the pKK009 DNA was removed by HindIII digestion, 33 different types of plasmids (pKK010-series plasmids) were obtained out of 58 clones tested, but no DNA rearrangement could be observed. On the basis of a comparison of the detailed restriction maps of these pKK010-series plasmids, we propose a model in which four DNA segments invert independently or in groups within the 1.95-kilobase region of R64, so that the arrangements of these four segments change randomly. The fixed pKK010-series plasmid DNA was again rearranged in the presence of R64, indicating that trans-acting gene function may be present to mediate the DNA rearrangement. The gene (tentatively designated as rci) was located on a 4.5-kilobase E9' fragment of R64.