Lars J. Brandén

A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA–NLS peptide. The PNA–NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA–NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA–PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.

Brutons tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N‐terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild‐type (wt) and also a gain‐of‐function mutant of Btk are redistributed following a wide range of receptor‐mediated stimuli through phosphatidylinositol 3‐kinase (PI 3‐K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3‐K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3‐K. A gain‐of‐function mutant of Btk was found to be a potent inducer of lamellipodia and / or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co‐localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17‐Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3‐K and certain members of the Rho‐family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

Expression profiling in transformed human B cells: influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays

We have used both Clontech AtlasTM Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein‐Barr virus (EBV)‐transformed B cells and genes regulated by Brutons tyrosine kinase (Btk). We compared EBV‐transformed wild‐type (WT) B cells from a healthy individual, WT1 and an X‐linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were ≥1.9‐fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down‐regulated in XLA2, indicating their biological role. Higher‐fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real‐time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell‐specific OCT binding factor‐1 (OBF‐1) were induced ≥1.9‐fold in both XLA1 and XLA2 analyzed by AtlasTM filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses.

Adding functional entities to plasmids

Non‐viral gene therapy constitutes an alternative to the more common use of viral‐mediated gene transfer. Most gene transfer methods using naked DNA are based upon non‐sequence‐specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence‐specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long‐term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo. Copyright

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones.

It has been shown previously that mutations in the cytoplasmic protein kinase, Brutons tyrosine kinase (BTK) lead to X‐linked agammaglobulinemia, an inherited primary immunodeficiency, thus making it a potential candidate for gene therapy.

150. Cooperative strand invasion of super-coiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.

PNA: a universal tool in molecular biology: Peptide Nucleic Acids: Protocols and Applications edited by P.E. Nielsen and M. Egholm

1999, Horizon Scientific Press. UK£59.99 hbk (x+266 pages)ISBN 1 898 48616 6This book is an upgraded guide to the properties and possibilities of the DNA mimic peptide nucleic acid (PNA), which is more complete than the ‘Users guide’ found on the PerSeptive Biosystems homepage, even though it has been recently revised (http://www.pebio.com/ds/pna/users.html). As stated in the preface, the focus of this book is on the actual applications and protocols relating to PNA. The neutrality of the backbone and the flexibility of adding functional units to the PNA oligonucleotide make it a formidable tool, and its wide range of applications are well illustrated in the spectrum of protocols included in the book.The introduction gives the reader a good overview of the properties of the PNA molecule, as well as some of its drawbacks (e.g. the solubility problem of purine-rich sequences). The subsequent chapters cover the synthesis of PNA and different PNA hybrids. They illustrate the development in the efforts being made to synthesize PNA in order for it to be as readily achievable as nucleic acid synthesis. The reader is given good, solid advice when it comes to the type of linker that should be used for the different types of PNA–DNA hybrids.Some aspects of the radiolabeling of PNA molecules by using protein-specific enzymes are discussed, as well as the possibilities of using the free amino-terminus for fluorescence labeling. Some of the applications reflect the thermodynamic properties of PNA, and this is exemplified in Chapter 3.1 by Holmen and Norden.The use of PNA as a probe for hybridization in different detection systems is clearly illustrated in Chapter 3.2. In addition, PNA-assisted rare cleavage is a good illustration of what PNA can be used for with regard to blocking enzyme function. The PNA–DNA–PNA hybrid shields the DNA site from methylases and therefore a non-methylated site can be created, making the targeted sequence sensitive to restriction-enzyme cleavage after PNA removal. The labeling of plasmids with different types of fluorescent molecules, such as rhodamine, has long been a problem if one has needed to combine this with expression from a reporter gene in the plasmid. Philip Felgner and his colleagues (Chapter 4.5) have solved this by using a specifically engineered site in the reporter plasmids, enabling a PNA clamp to bind to the plasmid. The clamp is labeled either with a fluorescent label or with biotin. This technique has made it possible to track the fate of plasmids in the cells at the same time as maintaining gene expression.In conclusion, this book is a necessary requirement for anyone wanting to delve into the field of PNA. It will also be an aid to developing the new genetic tool that PNA represents, thus enabling researchers to create new solutions to problems in nucleic acid detection and gene medicine.