Bacteriophage Lambda DNA Research Articles

Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments.

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

Rapid Fluorescence-Free Detection of DNA Loop-Mediated Isothermal Amplification on Bare ITO Surface Under EG-FET Scheme

An extended gate field effect transistor (EG-FET)-based chip with a precision temperature control system is developed for real-time and fluorescence-free loop-mediated isothermal amplification (LAMP) detection. A label-free, non-optical instant method was used to detect the hydrogen ions released during nucleic acid amplification, rather than indirect measurements. The system comprises an EG-FET sensor, a temperature sensor, a heating block, and a signal processing system. Industrial grade bare indium tin oxide (ITO) is firstly demonstrated as the sensing material for nucleic acid amplification detection, such that stable and high hydrogen ion detection performance of -80.1±0.03 mV/pH with R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> of 0.998 is achieved with the developed sensor. Moreover, a printed circuit board (PCB) with high thermal conductivity was used as the sensor base to provide a uniform temperature distribution during the reaction. The results show that the system maintains a stable LAMP assay temperature of 63°C with a variation of just 0.25°C. Moreover, in a pH-sensitive LAMP assay, the developed sensor achieves a limit-of-detection down to 10 genomic copies per reaction of lambda phage DNA within 17 minutes. The novel and performance iso-thermal EG-FET system is capable of real-time detecting the LAMP DNA amplification, and thus has significant potential for future biomedical applications.

Extremely early onset of nonlinear viscoelasticity in dynamic shear of ideally monodisperse DNA

AbstractThis work reports that the rheology of ideally monodisperse lambda () phage DNA solutions is extremely sensitive to the applied strain in dynamic oscillatory experiments. DNA exhibits nonlinearity at strains far smaller than what is observed in conventional synthetic polymers. However, it is found that polydisperse calf thymus DNA does not exhibit the extreme strain sensitivity. By mixing samples of monodisperse DNA with the polydisperse calf thymus DNA, we correlate the molecular weight distribution (or polydispersity) to the onset of nonlinear viscoelasticity. We demonstrate that the strain sensitivity weakens as the polydispersity index increases. This is the first work correlating the inception of nonlinear viscoelasticity in entangled polymer system such as lambda DNA solution to the polydispersity of the samples. The experimental data suggests that ideally monodisperse polymer systems are very sensitive to the applied strain and have a very early transition to the nonlinear regime. We conclude that nominally monodisperse synthetic polymers are not sufficiently monodispersed to exhibit the early onset.

Regulation of the phage lambda viral packaging motor's grip and DNA end-clamp mechanism

Many viruses utilize ATP-powered motors to package DNA into viral capsids. Here we use single DNA manipulation with optical tweezers and rapid solution exchange to interrogate the dynamics of DNA gripping by the phage lambda motor. Binding of poorly hydrolyzed ATP analogs results in persistent gripping and when slips occasionally occur, the release velocity is very slow, indicating significant friction between the motor and DNA. ADP induces more frequent transitions between gripping and faster slipping. Independent of nucleotides, an “end clamp” mechanism arrests slipping when the end of the DNA is about to exit the capsid. These features are qualitatively similar to those exhibited by the phage T4 motor, although there are some notable differences. Specifically, there are quantitative differences in the DNA grip/release kinetics and their dependence on nucleotides and applied force. But the most striking difference is that there are frequent long pauses during DNA exit from the capsid even in the absence of nucleotides. This pausing occurs at low capsid filling and does not increase with length of DNA packaged, indicating that it is not caused by jamming/knotting of the packaged DNA, but is attributable to fluctuation of apo-state motor subunits into a DNA gripping conformation. This finding supports our previous hypothesis that pauses which are frequently observed during packaging are caused by fluctuation of motor subunits into such a conformation. Overall, compared with T4, the lambda motor exhibits stronger interactions with DNA, which may be attributable to the small terminase subunit in the lambda packaging motor complex.

Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters

Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter ‘601’ arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration. Nucleosome clusters and their structural fluctuations were visualized in confined nanochannels. Force spectroscopy revealed not only similar mechanical properties of the TAD-sized fibers as shorter fibers but also large rupture events, consistent with breaking the interactions between distant clutches of nucleosomes. Though the arrays of native human DNA, lambda-phage and ‘601’ DNA featured minor differences in reconstitution yield and nucleosome stability, the fibers’ global structural and mechanical properties were similar, including the interactions between nucleosome clutches. These single-molecule experiments quantify the mechanical forces that stabilize large TAD-sized chromatin domains consisting of disordered, dynamically interacting nucleosome clutches and their effect on the condensation of large chromatin domains.

Assessment of Deoxyribonuclease Activity Using DNA Molecules Immobilized Between Microelectrodes

Serum deoxyribonuclease I (DNase I) can serve as a functional biomarker for the therapeutic monitoring of acute myocardial infarction and other diseases. Here, we demonstrate that the electrical properties of DNA molecules can be exploited to monitor enzymatic activity. A label-free DNA biosensor for the detection of DNase I activity was devised based on electrochemical impedance spectroscopy (EIS). Multiple lambda phage DNA molecules were immobilized between two electrodes in a polydimethylsiloxane reservoir. An equivalent circuit estimated from the EIS measurement was used to calculate the impedance of DNA molecules between the electrodes. DNase detection was then achieved by measuring the increase in impedance, after DNA cleavage by DNase I. This was assessed by the impedance-increase ratio, defined as Rafter/Rbefore (where Rbefore and Rafter represent the resistance between the electrode-immobilized DNA molecules before and after DNase I treatment, respectively). After treatment with DNase I at a concentration 10−2 unit/μL, a reproducible impedance-increase ratio of approximately 3.3 times was obtained, with a standard deviation of less than 20%. When DNase solutions of various concentrations were introduced, we succeeded in obtaining a definite correlation between DNase concentration and impedance-increase rate, within the range of 10−4 unit/μL to 10−1 unit/μL.

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent.

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO2 as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin-mediated drug-delivery of RGD-modified DLDH (DLDHRGD ), leading to apoptotic cell death. In these experiments, DLDHRGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA-binding capability of this enzyme. To explore this interaction under cell-free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel-shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA-binding. A Docking algorithm for prediction of protein-DNA complexes, Paradoc, identified a putative DNA binding site at the C-terminus of DLDH. Our finding that TiO2 -bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.

Graphene Oxide Modified Electrode for DNA Amperometric Detection Using Microfluidic Electrophoresis Chip

The amperometric technique of capillary electrophoresis (CE) is an electrochemical detection method with various advantages that is suitable for biosensors. The amperometric detection system can be easily integrated with the CE system without the need for complicated additional equipment for optical detection. For this reason, the size of the sensor can be reduced to micro-scale, also reducing the cost of the fabrication process, enabling this system to be used in point-of-care applications [1]. The optical method depends on the detection of the fluorescence intensity of the target sample. However, amperometric detection omits the pretreatment process of the sample, such as purification extraction of samples and fluorescent staining for detection [2]. These features allow the device system to be miniaturized and having a rapid response for applications like lab-on-a-chip [3]. Modified Au electrodes integrated with PDMS based microfluidic channels were proposed as an on-chip amperometric detection of CE for DNA sensors. The graphene oxide (GO)-modified Au electrodes were fabricated via photolithography process and drop-casting deposition. First, the deposited Au electrodes were patterned with positive photoresist (PR) using the photolithography method. Next, the graphene oxide was deposited 5 layers on the bare Au using drop-casting technique. Finally, the PR was dissolved by acetone and the unwanted GO was lifted-off. The surface characterization of this the GO-modified Au electrodes is shown in Fig. 1(a) by microscopy image. It was observed that the GO was deposited on Au electrodes following the desired pattern. This treatment amplifies the current signal of amperometric detection of the CE microchip. The performance of the CE amperometric detection device has been demonstrated by sensing an amplicon lambda phage DNA. Then, 1 µL of amplified lambda phage DNA (1 ng/µL) using the conventional polymerase chain reaction (PCR) with a 30-cycle reaction was used as target sample. Figure 1(b) shows the current peak of amperometric detection before (1.9 µA) and after (6.6 µA) GO-modified on the Au electrodes which amplified the sensitivity more than 3.5 times before electrode modification. The emphasized detection of lambda phage DNA using modified electrodes gives a further possibility of biomedical applications through our developed microchip.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

A Dual-Sensing Thermo-Chemical ISFET Array for DNA-Based Diagnostics.

This paper presents a 32×32 ISFET array with in-pixel dual-sensing and programmability targeted for on-chip DNA amplification detection. The pixel architecture provides thermal and chemical sensing by encoding temperature and ion activity in a single output PWM, modulating its frequency and its duty cycle respectively. Each pixel is composed of an ISFET-based differential linear OTA and a 2-stage sawtooth oscillator. The operating point and characteristic response of the pixel can be programmed, enabling trapped charge compensation and enhancing the versatility and adaptability of the architecture. Fabricated in 0.18 μm standard CMOS process, the system demonstrates a quadratic thermal response and a highly linear pH sensitivity, with a trapped charge compensation scheme able to calibrate 99.5% of the pixels in the target range, achieving a homogeneous response across the array. Furthermore, the sensing scheme is robust against process variations and can operate under various supply conditions. Finally, the architecture suitability for on-chip DNA amplification detection is proven by performing Loop-mediated Isothermal Amplification (LAMP) of phage lambda DNA, obtaining a time-to-positive of 7.71 minutes with results comparable to commercial qPCR instruments. This architecture represents the first in-pixel dual thermo-chemical sensing in ISFET arrays for Lab-on-a-Chip diagnostics.

3D control stretched length of lambda-phage WLC DNA molecule by nonlinear optical tweezers

In this paper, the general Langevin equations of motion for the polystyrene bead linked to the lambda-phage worm-like chain DNA molecule embedded in the fluid under the nonlinear optical tweezers is derived in 3D space. Using the finite difference method, the dynamical properties of the bead trapped by the nonlinear optical tweezers using a thin layer of Acid Blue 29 are numerically studied. Results in, the stretched length of the lambda-phage worm-like chain DNA molecule can be controlled in 3D space by finely tuning of the laser power.

Silver(I) complexes with 1,10-phenanthroline-based ligands: The influence of epoxide function on the complex structure and biological activity

In a continuing search for a novel metal-containing antimicrobial agents, the present study reports the synthesis, characterization and biological evaluation of two silver(I) complexes with 1,10-phenanthroline-based ligands, [Ag(1,10-phen)2]CF3COO·H2O (Ag1) and [Ag(CF3COO)(5,6-epoxy-1,10-phen)]2 (Ag2), 1,10-phen is 1,10-phenanthroline and 5,6-epoxy-1,10-phen is 5,6-epoxy-5,6-dihydro-1,10-phenanthroline. The complexes were characterized by different spectroscopic techniques (IR, 1H and 13C NMR and UV–Vis), while the crystal structure of Ag2 complex was determined by a single-crystal X-ray diffraction analysis. The spectroscopic data confirmed that the structure of Ag1 complex, with silver(I) ion tetrahedrally coordinated by two bidentate 1,10-phen ligands, is in accordance to the previous report (S.E. Paramonov et al., 2003). The crystallographic results showed that in dinuclear Ag2 complex, both Ag(I) ions are coordinated bidentately by 5,6-epoxy-1,10-phen and monodentately by trifluoroacetate, with presence of the short Ag∙∙∙Ag contact of 2.963 Å. Both silver(I) complexes were evaluated in vitro for antimicrobial activity against four bacterial and four Candida species, showing selectivity towards the investigated species of Candida with minimal inhibitory concentrations (MICs) between 0.9 and 12.5 µM. Moreover, Ag2 complex manifested significant antiproliferative properties in the case of a range of human cell lines, including human breast cancer (MDA-MB 231), which resulted from the presence of epoxy functional group in the ligand. The gel electrophoresis results obtained from the studies of Ag1 and Ag2 interactions with bacteriophage lambda DNA (λDNA) suggested that these complexes did not cause DNA degradation.

High levels of AAV vector integration into CRISPR-induced DNA breaks

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

3D Columnar Phase of Stacked Short DNA Organized by Coherent Membrane Undulations.

We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (dsDNA) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing dsDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA-DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent "crystal-like" undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large dsDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (∝ κh2/dsDNA4, h2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. The findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.

Stability of dry Phage Lambda DNA irradiated with swift heavy ions

Effects of irradiations of Phage Lambda dry DNA samples at room and cryogenic temperatures with 158 MeV Xe and 48 MeV Ar ions were investigated. These ions primary transfer energy into the electronic subsystem of a target: dE/dx = Se, SeXe = 10.8 keV/nm and SeAr = 3.5 keV/nm. Site-specific enzymes restriction endonucleases were applied to indicate DNA damages induced by these ions. Electrophoresis was applied to analyze the dependencies of the distributions of DNA fragments sizes on the ion fluence ranging from 108 cm−2 to 1010 cm−2.Electron paramagnetic resonance (EPR) technique was used to investigate damages of a dry DNA sample irradiated at the cryogenic temperature (140 K) with 158 MeV 13254Xe26+ ions up to the fluence 8.6·109cm−2. A poorly resolved signal centered at the g-factor value for the free electron g ≈ ge was detected, which probably results from a mixture of different-type radicals trapped in the DNA film. The total concentration of paramagnetic species in this sample was estimated to be 1.3 × 1019 spin/g.

Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases

Our laboratory is interested in DNA and RNA methylation in E. coli and T. brucei as little is known about this form of epigenetic regulation in microorganisms. One methyltransferase being studied at this time is a putative DNA methyltransferase (TbDmt) from Trypanosoma brucei. The exact function of TbDmt is unknown but the protein strongly resembles bacterial DNA methyltransferases such as DNA cytosine methyltransferase (EcDcm) from E. coli. To test our hypothesis that TbDmt is a DNA methyltransferase, we expressed TbDmt in bacteria and created two chimeric protein sequences switching the DNA binding domain and enzymatic domain of EcDcm and TbDmt. Exchanging the DNA binding domain and enzymatic domain of TbDmt with a known methyltransferase may help us discover the function of the enzyme and, if it is a methyltransferase, what DNA sequence is targeted for methylation. Plasmids were made containing the sequences for EcDcm, TbDmt, and both chimeric proteins where the genes are adjacent to the T5 promoter and lac operator. E. coli lacking a cytosine DNA methylation pathway were transformed with the plasmids and expression was induced with IPTG. All four proteins were produced at 20°C, but the proteins with the TbDmt DNA binding domain were less soluble than the other two. The plasmids were re‐isolated after three hours of growth in the presence of IPTG. The plasmids were then digested with eight restriction enzymes blocked by methylation. Each digestion was run on an agarose gel with DNA from an uninduced cell and unmethylated phage lambda DNA as controls. EcDcm methylated at its expected site, 5′CCWGG3′, as determined by resistance to PspGI. In addition, there is partial resistance to HpaII in cells containing EcDcm indicating methylation at 5′CCGG3′ or related sequences. We are currently testing restriction enzyme sensitivity in the samples containing full length TbDmt and chimeric TbDMTs. Further work will be done to improve the conditions in which the chimeric proteins are produced to enhance folding of the potential DNA methyltransferases. In summary, this work contributes to our limited knowledge of DNA methylation enzymes and targets in bacteria and protists.Support or Funding InformationNIH, 1R15AI133428‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Budding and Division of Giant Vesicles Linked to Phospholipid Production

The self-reproduction of supramolecular assemblies based on the synthesis and self-assembly of building blocks is a critical step towards the construction of chemical systems with autonomous, adaptive, and propagation properties. In this report, we demonstrate that giant vesicles can grow and produce daughter vesicles by synthesizing and incorporating phospholipids in situ from ad-hoc precursors. Our model involves acyl chain elongation via copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition reaction and the ensuing production of synthetic phospholipids to induce budding and division. In addition, the growth and budding of giant vesicles were compatible with the encapsulation and transfer of macromolecules as large as lambda phage DNA to the buds. This chemical system provides a useful model towards the implementation of cell-like compartments capable of propagation and transport of biological materials.

Phytochemical profiling of Artocarpus lakoocha Roxb. leaf methanol extract and its antioxidant, antimicrobial and antioxidative activities

Objective: To explore the phytochemical profile of Artocarpus lakoocha Roxb. leaves both qualitatively and quantitatively, and validate its role as a potent antioxidant and antimicrobial agent. Methods: Extraction and isolation of different compounds were done from the leaves of Artocarpus lakoocha based on solvent fractionation method. Subsequently, quantitative and qualitative phytochemical profiling along with antioxidant, antimicrobial and antioxidative activities were tested following standard protocols. Results: Among the five fractions, methanol fraction of Artocarpus lakoocha exhibited higher content of phytochemical compounds [phenols = (3 175.21 ±290.43) mg GAE/g dry extract, flavonoids = (1 173.15±47.52) mg QE/g dry extract and tannins = (923.53±95.21) mg TAE/ g dry extract] as compared to other fractions. The methanol fraction showed the highest antioxidant activity in DPPH and ABTS radical scavenging assays with IC50 of (111.98±34.20) μg/mL and (138.26±0.66) μg/mL, respectively, and the best reduction potential with a value of (316.81±2.96) mg QE/g dry extract in reducing power assay. There was significant correlation between the amount of phytochemicals and antioxidant activities. Moreover, the extract successfully protected Lambda phage DNA from damage at 5 and 6 mg/mL concentration and exhibited substantial bactericidal as well as fungicidal activity. The GC-MS analysis of methanol fraction of Artocarpus lakoocha revealed diethyl phthalate as the main phytochemical compound, along with 3,4-dihydroxymandelic acid, 9-octyl eicosane and 7,8-didehydro-3- methoxy-17-methyl-6-methylene morphinan. Conclusions: The methanol fraction of Artocarpus lakoocha could be used as a potent antioxidant and antimicrobial agent for sustainable agriculture and pharmaceutical purposes.

Carbohydrate Transport by Group Translocation: The Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System.

The Bacterial Phosphoenolpyruvate (PEP) : Sugar Phosphotransferase System (PTS) mediates the uptake and phosphorylation of carbohydrates, and controls the carbon- and nitrogen metabolism in response to the availability of sugars. PTS occur in eubacteria and in a few archaebacteria but not in animals and plants. All PTS comprise two cytoplasmic phosphotransferase proteins (EI and HPr) and a species-dependent, variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC, IID). EI and HPr transfer phosphorylgroups from PEP to the IIA units. Cytoplasmic IIA and IIB units sequentially transfer phosphates to the sugar, which is transported by the IIC and IICIID integral membrane protein complexes. Phosphorylation by IIB and translocation by IIC(IID) are tightly coupled. The IIC(IID) sugar transporters of the PTS are in the focus of this review. There are four structurally different PTS transporter superfamilies (glucose, glucitol, ascorbate, mannose) . Crystal structures are available for transporters of two superfamilies: bcIICmal (MalT, 5IWS, 6BVG) and bcIICchb (ChbC, 3QNQ) of B. subtilis from the glucose family, and IICasc (UlaA, 4RP9, 5ZOV) of E. coli from the ascorbate superfamily . They are homodimers and each protomer has an independent transport pathway which functions by an elevator-type alternating-access mechanism. bcIICmal and bcIICchb have the same fold, IICasc has a completely different fold. Biochemical and biophysical data accumulated in the past with the transporters for mannitol (IICBAmtl) and glucose (IICBglc) are reviewed and discussed in the context of the bcIICmal crystal structures. The transporters of the mannose superfamily are dimers of protomers consisting of a IIC and a IID protein chain. The crystal structure is not known and the topology difficult to predict. Biochemical data indicate that the IICIID complex employs a different transport mechanism . Species specific IICIID serve as a gateway for the penetration of bacteriophage lambda DNA across, and insertion of class IIa bacteriocins into the inner membrane. PTS transporters are inserted into the membrane by SecYEG translocon and have specific lipid requirements. Immunoelectron- and fluorescence microscopy indicate a non-random distribution and supramolecular complexes of PTS proteins.

Synthetic DNA fragments bearing ICR cis elements become differentially methylated and recapitulate genomic imprinting in transgenic mice

BackgroundGenomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5′ fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation.ResultsTo ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5′ sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question.ConclusionsOur results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo.