GC-rich DNA Research Articles

Egr-1 deficiency protects host against Pseudomonas aeruginosa lung infection

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of nosocomial infections in cystic fibrosis patients and immunocompromised individuals. The molecular mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Early Growth Response 1 (Egr-1) is a zinc-finger transcription factor that binds to the GC-rich DNA consensus sequences in the promotor of target genes, important for cell growth, differentiation and survive. Aberrant expression of Egr-1 has been implicated in many inflammatory diseases. In this study, we demonstrate that Egr-1 deficiency protects host against P. aeruginosa infection in a mouse model of acute bacterial pneumonia. Egr-1 expression was rapidly and transiently induced both in vitro and in vivo upon P. aeruginosa infection. Egr-1-deficient mice displayed decreased disease score, reduced systemic levels of proinflammatory cytokines and impaired NF-κB and NFAT activation compared to wild-type mice. Interestingly, Egr-1 deficiency leads to enhanced bacterial clearance and increased nitric oxide production in lung whereas it has no impact on neutrophil recruitment. Further studies revealed that Egr-1-deficient neutrophils displayed elevated bacterial killing ability. Altogether, these findings suggest that Egr-1 promotes inflammatory responses by enhancing NF-κB and NFAT activation and plays a detrimental role in host defense against P. aeruginosa lung infection by negatively regulating nitric oxide production for bacterial clearance.

Cytogenetic Studies in Glossogobius giuris (Hamilton, 1822) Through NOR-Staining and FISH

The Glossogobius giuris (Gobiidae), tank goby—a species of goby fish, is native mainly to freshwater and estuaries and has importance in the aquarium trade. It is classified as least concern under IUCN Red List. Live individuals of this species were collected from Loktak Lake in Manipur for cytogenetic investigation to reveal the nucleolar organizer region (NOR) through silver nitrate (AgNO3) and chromomycin A3 (CMA3) staining as well as DAPI staining and single-color fluorescence in situ hybridization (FISH) using 18S rDNA sequence as probe. Analysis of more than 50 metaphase chromosome complement (obtained from colchicine–potassium chloride–Carnoy’s fixation–Giemsa staining procedures) showed the presence of 46 diploid chromosome numbers with all telocentric having fundamental arm number as 46 without any heteromorphic pair. One pairs of silver-stained NORs, situated terminally on the telocentric chromosome, were observed. Similarly, one pair of CMA3-positive sites were observed on the chromosome that suggested abundance of GC-rich repetitive DNAs in this region. One pair of 18S rDNA positive sites was observed on the telocentric chromosomes using FISH. These karyological features can be useful markers in cytotaxonomy and conservation of this species.

Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell.

Objective Easily oxidizable GC-rich DNA (GC-DNA) fragments accumulate in the cell-free DNA (cfDNA) of patients with various diseases. The human oxidized DNA penetrates the MCF7 breast cancer cells and significantly changes their physiology. It can be assumed that readily oxidizable GC-DNA fragments can penetrate the cancer cells and be expressed. Methods MCF7 cells were cultured in the presence of two types of GC-DNA probes: (1) vectors pBR322 and pEGFP and (2) plasmids carrying inserted human rDNA (pBR322-rDNA and pEGFP-rDNA). pEGFP and pEGFP-rDNA contained a CMV promoter and a fluorescent protein gene EGFP. ROS generation rate, accumulation of the DNA probes in MCF7, 8-oxodG content, expression of EGFP and NOX4, and localization of EGFP, NOX4, and 8-oxodG in MCF7 were explored. The applied methods were qPCR, fluorescent microscopy (FM), immunoassay, and flow cytometry (FCA). Results When GC-DNA is added to the cell culture medium, it interacts with the cell surface. At the site of GC-DNA contact with the cell, NOX4 is expressed, and ROS level increases. The ROS oxidize the GC-DNA. When using the plasmids pEGFP and pEGFP-rDNA, an increase in the amount of the DNA EGFP, RNA EGFP, and EGFP proteins was detected in the cells. These facts suggest that GC-DNA penetrates the cells and the EGFP gene is expressed. Insertions of the rDNA significantly increase the GC-DNA oxidation degree as well as the rate of plasmid transfection into the cells and the EGFP expression level. In the nucleus, the oxidized GC-rDNA fragments, but not the vectors, are localized within the nucleolus. Conclusions GC-rich cfDNA fragments that are prone to oxidation can easily penetrate the cancer cells and be expressed. The cfDNA should become a target for the antitumor therapy.

A single species, two basic chromosomal numbers: case of Lygeum spartum (Poaceae)

The existence of two chromosome numbers (2n = 16 and 2n = 40) in Lygeum spartum is confirmed in the Algerian steppe populations of Oran region. Chromosome counts were established for 11 Algerian populations and three supplementary populations from Italy, Spain and Greece. The karyotypes were characterized by chromosome markers obtained using fluorescence in situ hybridization and fluorochrome bandings. The organization of 5S and 18S-5.8S-26S (35S) rDNA was studied in both cytotypes. Six signals of 35S and 2 signals of 5S were observed in 2n = 16 population, while 10 signals of 35S and 4 signals of 5S were detected in the population with 2n = 40. All 35S loci were also strongly marked by chromomycin, but negatively stained by Hoechst, which indicates the presence of GC rich DNA in rDNA regions. The B chromosomes were found in both cytotypes, bearing a 35S locus in 2n = 16 population. Genome size, determined by cytometry of 10 populations, ranged from 9.27 pg for 2n = 16 to 26.63 pg for 2n = 40 populations. The sequencing of plastid and nuclear DNA markers did not reveal major differences among 2n = 16 and 2n = 40 populations. However, given the differences between two cytotypes and based on their morphological and cytogenetic characteristics, the 2n = 16 cytotype merits novel taxonomic treatment.

Phenylboronic acid-modified polyamidoamine-mediated delivery of short GC rich DNA for hepatocarcinoma gene therapy.

Phenylboronic acid was introduced on the surface of polyamidoamine to construct a derivative PP, which was further used as a tumor-targeting carrier for realizing the delivery of short GC rich DNA (GCD). The PP-mediated GCD delivery could disrupt the polymerization of microtubules and thus trigger a strong anti-proliferative effect through the induction of cell apoptosis and cell cycle arrest, using hepatocellular carcinoma cell line HepG2 as a model. In addition, the transfection of PP/GCD nanoparticles could efficiently suppress cell migration and invasion. Moreover, the intravenous injection of PP/GCD nanoparticles could dramatically decrease the tumor growth by inducing the in situ apoptosis of the tumor and meanwhile it exhibited a desirable safety profile. Overall, the development of tumor-targeting carrier PP will provide a promising platform for GCD delivery to obtain an anti-cancer efficacy which is beneficial for facilitating tumor gene therapy in future clinical applications.

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass.

The DNA replisome inevitably encounters DNA damage during DNA replication. The T7 DNA replisome contains a DNA polymerase (gp5), the processivity factor thioredoxin (trx), a helicase-primase (gp4), and a ssDNA-binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated strand-displacement DNA synthesis past 8-oxoG or O6 -MeG lesions at the synthetic DNA fork by the T7 DNA replisome. DNA damage does not obviously affect the binding affinities between helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6 -MeG-as well as GC-rich template sequence clusters-inhibit strand-displacement DNA synthesis and produce partial extension products. Relative to the gp4 ΔC-tail, gp4 promotes DNA damage bypass. The presence of gp2.5 also promotes it. Thus, the interactions of polymerase with helicase and ssDNA-binding protein facilitate DNA damage bypass. Accessory proteins in other complicated DNA replisomes also facilitate bypassing DNA damage in similar manner. This work provides new mechanistic information relating to DNA damage bypass by the DNA replisome.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic THP-1 cells.

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing

The thermostable Geobacillus stearothermophilus GsI–IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI–IIC. We found that GsI–IIC, like other group IIC introns, retrohomes into sites having a 5′-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7–19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5′-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5′-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI–IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.

Smart Magnetic Nanoaptamer: Construction, Subcellular Distribution, and Silencing HIF for Cancer Gene Therapy.

Attenuating the expression of HIF-1α (hypoxic inducible factor) by siRNA has an effect on the proliferation of hypoxia cancers. Mitochondria targeting siRNA may silence the level of HIF-1α for cancer gene therapy. A GAG-rich DNA was conjugated to GC-rich DNA for the synthesis of functional magnetic nanoaptamer (DNA-Fe3O4) to keep the innate character of the targeting aptamer. The DNA-Fe3O4 can load the hydrophobic dye (BODIPY-OCH3) by the GC-rich sequences, resulting in fluorescent nanoaptamer (BFe@DNA). Self-assembly of BFe@DNA with target aptamer resulted in the formation of BFe@DNAH. Subcellular fluorescence imaging results confirm that BFe@DNAH can accumulate in MCF-7 cells and selectively target mitochondrion. In particular, BFe@DNAH can transport siRNA to breast cancer cells or tissues for the attenuation of HIF-1α and ATP and the inhibition on growth of cancer cells in vivo. Therefore, BFe@DNAH is a smart nanoaptamer platform for the development of subcellular imaging agents and gene therapy.

Ehrlichia chaffeensis TRP120 nucleomodulin binds DNA with disordered tandem repeat domain.

Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, secretes several effector proteins that bind host DNA to modulate host gene expression. The tandem repeat protein 120 (TRP120), one of the largest effector proteins, has four nearly identical tandem repeat (TR) regions that each consists of 80 amino acids. In addition to playing a role in ehrlichial binding and internalization, TRP120 translocates to the host nucleus where it is thought to function as a transcription factor that modulates gene expression. However, sequence analysis of TRP120 does not identify the presence of DNA-binding or trans-activation domains typical of classical eukaryotic transcription factors. Thus, the mechanism by which TRP120 binds DNA and modulates gene expression remains elusive. Herein, we expressed the TR regions of the TRP120 protein, and characterized its solution structure and ability to bind DNA. TRP120, expressed as either a one or two TR repeat, is a monomer in solution, and is mostly disordered as determined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy, we further show that the 1 TR construct selectively binds GC-rich DNA. Although low pH was required for TRP120 TR-DNA interaction, acidic pH alone does not induce any significant structural changes in the TR region. This suggests that TRP120 folds into an ordered structure upon forming a protein-DNA complex, and thus folding of TRP120 TR is coupled with DNA binding.

Divalent cations are dispensable for binding to DNA of a novel positively charged olivomycin A derivative.

The current model of binding of the antitumor antibiotic olivomycin A (1) to GC-rich DNA regions presumes that coordination of the magnesium divalent cation with drug dimers is necessary for binding of 1 into the minor groove of the DNA duplex. Previously we have synthesized the derivatives of 1 termed ‘short acid’ (2) and its N,N-dimethylaminoethylamide (3). The latter compound demonstrated an improved tolerance in vivo compared to 1 and good therapeutic potency in animal models. We herein report that compound 3 is able to form stable complexes with DNA in the absence of Mg2+, in striking contrast to 1 whose binding to the DNA absolutely requires Mg2+. The mode of binding of 3 to DNA is similar in the presence or absence of Mg2+ as determined by circular dichroism. The affinity to DNA of 3 in Mg2+-free solution was similar to that of 1 or 3 in the presence of Mg2+ at low ionic strength. Non-electrostatic contributions to total free energy of binding of 1 and 3 to DNA were comparable for Mg2+-free complexes. Our data strongly suggest that electrostatic interaction of the positively charged 3 can compensate for the absence of divalent ions in complexes with DNA. This new property of the olivomycin A derivative expands the mechanistic knowledge of the modes of interaction with DNA of small molecular weight drug candidates.

Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells

Platforms for targeted drug-delivery must simultaneously exhibit serum stability, efficient directed cell internalization, and triggered drug release. Here, using lipid-mediated self-assembly of aptamers, we combine multiple structural motifs into a single nanoconstruct that targets hepatocyte growth factor receptor (cMet). The nanocarrier consists of lipidated versions of a cMet-binding aptamer and a separate lipidated GC-rich DNA hairpin motif loaded with intercalated doxorubicin. Multiple 2′,6′-dimethylazobenzene moieties are incorporated into the doxorubicin-binding motif to trigger the release of the chemotherapeutics by photoisomerization. The lipidated DNA scaffolds self-assemble into spherical hybrid-nanoconstructs that specifically bind cMet. The combined features of the nanocarriers increase serum nuclease resistance, favor their import into cells presumably mediated by endocytosis, and allow selective photo-release of the chemotherapeutic into the targeted cells. cMet-expressing H1838 tumor cells specifically internalize drug-loaded nanoconstructs, and subsequent UV exposure enhances cell mortality. This modular approach thus paves the way for novel classes of powerful aptamer-based therapeutics.

How abasic sites impact hole transfer dynamics in GC-rich DNA sequences.

Changes in DNA charge transfer properties upon the creation of apurinic and apyrimidinic sites have been used to monitor DNA repair processes, given that such lesions generally reduce charge transfer yields. However, because these lesions translate into distinct intra and extrahelical conformations depending on the nature of the unpaired base and its DNA context, it is unclear the actual impact of such diverse conformations on charge transfer. Here we combine classical molecular dynamics, quantum/molecular mechanics (QM/MM) calculations, and kinetic Monte Carlo simulations to investigate the impact of abasic sites on the structure and hole transfer (HT) properties of DNA. We consider both apurinic and apyrimidinic sites in polyG and polyGC sequences and find that most situations lead to intrahelical conformations where HT rates are significantly slowed down due to the energetic disorder induced by the abasic void. In contrast, the presence of an unpaired C flanked by C bases leads to an extrahelical conformation where stacking among G sites is reduced, leading to an attenuation of electronic couplings and a destabilization of hole states. Interestingly, this leads to an asymmetric HT behavior, given that the 5' to 3' transfer along the G strand is slowed down by one order of magnitude while the opposite 3' to 5' transfer remains similar to that estimated for the reference polyG sequence. Our simulations thus suggest that electrochemical monitoring of the DNA repair process following changes in charge transfer properties can miss repair events linked to abasic sites adopting extrahelical conformations.

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis.

Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format.

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

Characterization and genomic study of \u201cphiKMV-Like\u201d phage PAXYB1 infecting Pseudomonas aeruginosa

Bacteriophage PAXYB1 was recently isolated from wastewater samples. This phage was chosen based on its lytic properties against clinical isolates of Pseudomonas aeruginosa (P. aeruginosa). In the present study, characterized PAXYB1, clarified its morphological and lytic properties, and analyzed its complete genome sequence. Based on the morphology of PAXYB1, it is a Podoviridae. The linear GC-rich (62.29%) double-stranded DNA genome of PAXYB1 is 43,337 bp including direct terminal repeats (DTRs) of 468 bp. It contains 60 open reading frames (ORFs) that are all encoded within the same strand. We also showed that PAXYB1 is a virulent phage and a new member of the phiKMV-like phages genus. Twenty-eight out of sixty predicted gene products (gps) showed significant homology to proteins of known function, which were confirmed by analyzing the structural proteome. Altogether, our work identified a novel lytic bacteriophage that lyses P. aeruginosa PAO1 and efficiently infects and kills several clinical isolates of P. aeruginosa. This phage has potential for development as a biological disinfectant to control P. aeruginosa infections.

ZBP-89 function in colonic stem cells and during butyrate-induced senescence.

ZBP-89 (Zfp148, ZNF148) is a Kruppel-type zinc-finger family transcription factor that binds to GC-rich DNA elements. Earlier studies in cell lines demonstrated that ZBP-89 cooperates with Wnt β-catenin signaling by inducing β-catenin gene expression. Since β-catenin levels are normally highest at the crypt base, we examined whether ZBP-89 is required for stem cell maintenance. Lineage-tracing using a Zfp148CreERT2 transgenic line demonstrated expression in both intestine and colonic stem cells. Deleting the Zfp148 locus in the colon using the Cdx2NLSCreERT2 transgene, reduced the size and number of polyps formed in the Apc-deleted mice. Since colon polyps form in the presence of butyrate, a short chain fatty acid that suppresses cell growth, we examined the direct effect of butyrate on colon organoid survival. Butyrate induced senescence of colon organoids carrying the Apc deletion, only when Zfp148 was deleted. Using quantitative PCR and chromatin immunoprecipitation, we determined that butyrate treatment of colon cell lines suppressed ZNF148 gene expression, inducing CDKN2a (p16Ink4a) gene expression. Collectively, Zfp148 mRNA is expressed in CBCs, and is required for stem cell maintenance and colonic transformation. Butyrate induces colonic cell senescence in part through suppression of ZBP-89 gene expression and its subsequent occupancy of the CDKN2A promoter.

Experimental Probing and Molecular Dynamics Simulation of the Molecular Recognition of DNA Duplexes by the Flavonoid Luteolin.

Luteolin (C15H10O6) is an important flavonoid found in many fruits, plants, medicinal herbs, and vegetables exhibiting many pharmacological properties. The anticancer, antitumor, antioxidant, and anti-inflammatory activities of luteolin have been reported. The pharmacological action of small molecules is dependent upon its interaction with biomacromolecules. The interactions of small molecules with DNA play a major role in the transcription and translation process. In this work, we explored the energetic profile of DNA-luteolin interaction by isothermal titration calorimetry (ITC). The effect of temperature and salt concentration on DNA binding was examined by UV-Vis method. The mode of interaction was further probed by UV melting temperature analysis and differential scanning calorimetry. An atomic level insight on the recognition of luteolin with DNA was achieved by employing molecular dynamics (MD) simulation on luteolin in complex with AT- and GC-rich DNA sequences. AMBER force field proves to be appropriate in providing an understanding on the binding mode and specificity of luteolin with duplex DNA. MD results suggest a minor groove binding of luteolin with DNA and the binding free energy obtained is in agreement with the experimental results.

Evolutionary implications of heterochromatin and rDNA in chromosome number and genome size changes during dysploidy: A case study in Reichardia genus.

In this study we showed that constitutive heterochromatin, GC-rich DNA and rDNA are implicated in chromosomal rearrangements during the basic chromosome number changing (dysploidy) in Reichardia genus. This small Mediterranean genus comprises 8–10 species and presents three basic chromosome numbers (x = 9, 8 and 7). To assess genome evolution and differentiation processes, studies were conducted in a dysploid series of six species: R. dichotoma, R. macrophylla and R. albanica (2n = 18), R. tingitana and R. gaditana (2n = 16), and R. picroides (2n = 14). The molecular phylogeny reconstruction comprised three additional species (R. crystallina and R. ligulata, 2n = 16 and R. intermedia, 2n = 14). Our results indicate that the way of dysploidy is descending. During this process, a positive correlation was observed between chromosome number and genome size, rDNA loci number and pollen size, although only the correlation between chromosome number and genome size is still recovered significant once considering the phylogenetic effect. Fluorescent in situ hybridisation also evidenced changes in number, position and organisation of two rDNA families (35S and 5S), including the reduction of loci number and, consequently, reduction in the number of secondary constrictions and nuclear organising regions from three to one per diploid genome. The potential mechanisms of chromosomal and genome evolution, strongly implicating heterochromatin, are proposed and discussed, with particular consideration for Reichardia genus.

A High-Throughput PCR-Amplification of GC-Rich DNA Sequences

A High-Throughput PCR-Amplification of GC-Rich DNA Sequences