Molecular identification of individual oak and fir trees from maternal tissues of their fruits or seeds

The applicability of DNA markers to purely maternal tissues has been scarcely addressed in trees. We have focused on non-parenchymatic maternal tissues of the fruits and seeds of pedunculate oak (Quercus robur L.) and silver fir (Abies alba Mill.) and investigated whether they can be used for a direct molecular identification of the mother trees. Total DNA with sufficient quantity and quality was extracted from single woody pericarps of acorns as well as from single dry wings of silver fir seeds. The DNA was analysed by PCR at highly polymorphic microsatellite loci. A comparison of the multi-locus genotypes from pericarps and wings with those of the respective mother trees revealed absolute identity. Thus, mother trees could be identified by genotyping their fruits or seeds. The results demonstrate the applicability of DNA fingerprinting to woody and/or dry seed tissues without the destruction of embryos and endosperm or a significant contamination. Progress is now expected in dispersal biology as well as in forensics and forest management.     

A simple and rapid protein array based method for the simultaneous detection of biowarfare agents

A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap and easy to handle technology solution that combines a microtube-integrated protein chip with the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized. All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as low as in well established ELISAs. Most interesting, as a result of careful antibody screening and testing, it is currently possible to analyse at least five of the "dirty dozen" agents on one single protein chip in parallel.     

Cation mediation of radical transfer between Trp48 and Tyr356 during O2 activation by protein R2 of Escherichia coli ribonucleotide reductase: relevance to R1-R2 radical transfer in nucleotide reduction?

A tryptophan 48 cation radical (W48(+)(*)) forms concomitantly with the Fe(2)(III/IV) cluster, X, during activation of oxygen for tyrosyl radical (Y122.) production in the R2 subunit of class I ribonucleotide reductase (RNR) from Escherichia coli. W48(+)(*) is also likely to be an intermediate in the long-range radical transfer between R2 and its partner subunit, R1, during nucleotide reduction by the RNR holoenzyme. The kinetics of decay of W48(+)(*) and formation of tyrosyl radicals during O(2) activation (in the absence of R1) in wild-type (wt) R2 and in variants with either Y122, Y356 (the residue thought to propagate the radical from W48(+)(*) into R1 during turnover), or both replaced by phenylalanine (F) have revealed that the presence of divalent cations at concentrations similar to the [Mg(2+)] employed in the standard RNR assay (15 mM) mediates a rapid radical-transfer equilibrium between W48 and Y356. Cation-mediated propagation of the radical from W48 to Y356 gives rise to a fast phase of Y. production that is essentially coincident with W48(+)(*) formation and creates an efficient pathway for decay of W48(+)(*). Possible mechanisms of this cation mediation and its potential relevance to intersubunit radical transfer during nucleotide reduction are considered.     

Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc-Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the alpha6 and beta1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-beta. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of beta1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc-Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment.     

Prion protein-related proteins from zebrafish are complex glycosylated and contain a glycosylphosphatidylinositol anchor

A hallmark of prion diseases in mammals is a conformational transition of the cellular prion protein (PrP(C)) into a pathogenic isoform termed PrP(Sc). PrP(C) is highly conserved in mammals, moreover, genes of PrP-related proteins have been recently identified in fish. While there is only little sequence homology to mammalian PrP, PrP-related fish proteins were predicted to be modified with N-linked glycans and a C-terminal glycosylphosphatidylinositol (GPI) anchor. We biochemically characterized two PrP-related proteins from zebrafish in cultured cells and show that both zePrP1 and zeSho2 are imported into the endoplasmic reticulum and are post-translationally modified with complex glycans and a C-terminal GPI anchor.     

Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events

The hepatitis B virus (HBV) is an enveloped DNA virus which is highly infectious in vivo. In vitro, only primary hepatocytes of humans and Tupaia belangeri or the novel HepaRG cell line are susceptible to HBV, but infection is inefficient and study of early infection events in single cells is unsatisfactory. Since hepatoma cells replicate the virus efficiently after transfection, this limited infection efficiency must be related to the initial entry phase. Here, we describe the lipid-based delivery of HBV capsids into nonsusceptible cells, circumventing the natural entry pathway. Successful infection was monitored by observing the emergence of the nuclear viral covalently closed circular DNA and the production of progeny virus and subviral particles. Lipid-mediated transfer initiated productive infection that was at least 100-fold more effective than infection of permissive cell cultures. High-dose capsid transfer showed that the uptake was not receptor limited and allowed the intracellular transport of capsids and genomes to be examined microscopically. The addition of inhibitors confirmed an entry pathway by fusion of the lipid with the plasma membrane. By indirect immune fluorescence and native fluorescence in situ hybridization, we followed the pathway of capsids and viral genomes in individual cells. We observed an active microtubule-dependent capsid transfer to the nucleus and a subsequent release of the viral genomes exclusively into the karyoplasm. Lipid-mediated transfer of viral capsids thus appears to allow efficient introduction of genetic information into target cells, facilitating studies of early infection events which are otherwise impeded by the small number of viruses entering the cell.     

Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated.     

Replication of Vibrio cholerae Chromosome I in Escherichia coli: Dependence on Dam Methylation

We successfully substituted Escherichia coli''s origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI_Vc). Replication from oriCI_Vc initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI_Vc were similar. Since Hda is involved in the conversion of DnaA^ATP (DnaA bound to ATP) to DnaA^ADP (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI_Vc allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI_Vc. We show that when E. coli''s origin of replication is substituted by oriCI_Vc, dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI_Vc in E. coli. In this model, methylation at oriCI_Vc would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.The genomes of Vibrio cholerae and several related Vibrio spp. are distributed between two circular chromosomes. Characterization of the origins of replication of V. cholerae chromosomes I and II (oriCI_Vc and oriCII_Vc, respectively) has shown that oriCI_Vc is similar to the origin of replication of the Escherichia coli chromosome, oriC, whereas oriCII_Vc is completely different (20). Like oriC, oriCI_Vc has five R-type DnaA boxes (53) as well as boxes conforming to the I and τ types (52, 61), and the DnaA protein is the rate-limiting factor in the initiation of replication in both cases (18). In E. coli, DnaA associates with both ATP and ADP, and the ATP-bound form is absolutely required for initiation to take place (reviewed in reference 60). When reaching a critical level, DnaA^ATP (DnaA bound to ATP) protein is proposed to form a helical filament, anchored at one or more R-boxes (54, 69), in which origin DNA wraps around the outside of the DnaA core (21) or where the DnaA wraps around oriC (61). In both cases, the topology of the DnaA-oriC nucleoprotein complex leads to formation of compensatory negative supercoiling that facilitates unwinding of the adjacent AT-rich region resulting in initiation. In both models, DnaA^ATP is absolutely required for initiation, and in agreement with this, DnaA^ATP was found to be the rate-limiting factor for initiation in vivo (69).The V. cholerae oriCI_Vc also resembles oriC in having many potential sites for methylation by DNA adenine methyltransferase (Dam), although the number and position of the GATC sites differ slightly (see Fig. ​Fig.1).1). The role of Dam in initiation of chromosome replication has been studied mainly in E. coli. After initiation of DNA replication has occurred on a fully methylated oriC, the newly replicated hemimethylated origins are sequestered from the Dam methyltransferase and from reinitiation for approximately one-third of a doubling time. During this time interval, the activity and amount of DnaA available for initiation are reduced to prevent immediate reinitiation (reviewed in references 57 and 83). The sequestration is carried out by the SeqA protein that binds hemimethylated oriC GATC sequences with high affinity (48). In the absence of Dam methylation or SeqA, the same origin can be reinitiated in the same cell cycle, and initiations become asynchronous (9, 48).Open in a separate windowFIG. 1.Alignment of the E. coli minimal oriC with the corresponding region from V. cholerae chromosome I. The AT-rich sequence and the three 13-mer repeats L, M, and R found in E. coli (5) are indicated above the alignment. The 6-mer (A/T)GATCT boxes (80) are underlined. Other DnaA binding sites, i.e., R-boxes (53), I-boxes (52), and τ-boxes (61), are shown as boxed regions. Dam methylation sites (GATC) are shaded gray. The experimentally defined binding sites for integration host factor (IHF) (22) and factor for inversion stimulation (FIS) (65) in E. coli are indicated, and bases that match the consensus sequence are in boldface type. The single base difference between oriCI_Vc and oriCI_Vc* (see Materials and Methods) in the minimal origin region is shown below the two sequences. A gap introduced to maximize alignment of the two sequences is indicated by a dash in the sequence. Nucleotides that are identical in the two sequences are indicated by an asterisk below the two sequences.Genes encoding a Dam homologue and a SeqA homologue are present on Vibrio genomes, but there appear to be some differences between the functions of the proteins in E. coli and V. cholerae. dam has been found to be an essential gene in V. cholerae (33, 15), which is not the case in E. coli (48, 51). Conflicting data exist concerning the essentiality of seqA in V. cholerae (15, 72). The roles of Dam and SeqA in oriCI_Vc replication have been studied using minichromosomes, i.e., plasmids replicating exclusively from a cloned copy of oriCI_Vc (20). oriCI_Vc-based minichromosomes can replicate in wild-type E. coli cells but were unable to replicate in dam, seqA, and seqA dam mutants (20). The extrachromosomal existence of minichromosomes is dependent on their ability to initiate replication in synchrony with the chromosomal origin (46, 75). In E. coli cells mutated in dam or seqA, incompatibility exists between the oriC carried on minichromosomes and that of the chromosome due to origin competition (13), and when minichromosomes are maintained under selective pressure, they integrate into the origin region of the host chromosome (46, 75). Minichromosomes based on oriCI_Vc may also compete with the E. coli oriC for initiations in dam or seqA mutant cells. However, due to limited sequence identity, they may not be able to integrate into the E. coli chromosome. This could provide an explanation for the failure to introduce oriCI_Vc minichromosomes into dam and seqA mutant cells (20). Both dam and seqA genes could therefore be required for viability of V. cholerae for reasons not related to chromosome replication. In addition to its role in DNA replication, roles for Dam methylation in gene regulation and DNA repair have also been demonstrated in a number of bacteria (for reviews, see references 11, 45, 47, and 50). For V. cholerae as well as for Salmonella spp. and Yersinia pseudotuberculosis, Dam plays a role in virulence possibly through regulation of virulence gene expression (33). Less is known about the functions of seqA apart from its role in E. coli replication, but it has been suggested that SeqA functions as a nucleoid-organizing protein (for a review, see reference 83), and the E. coli chromosome has been demonstrated to have increased supercoiling in a seqA strain (85).Here we describe the first in vivo evidence that Dam plays an important role in the initiation of replication by facilitating the replication initiation at oriCI_Vc in E. coli. In addition, we show that SeqA does not carry an essential role in the initiation of replication.