Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I): effects of single base pair transitions on the cleavage of preferred pyrimidine-purine-pyrimidine triplets

The cleavage of DNA restriction fragments by bis(1,10-phenanthroline)copper(I) [[(OP)2CuI]+] is sequence dependent: the trimer TAT is most strongly preferred, while the trimer TGT and tetramers TAAT, TAGT, and CAGT are strongly to moderately preferred [Veal, J. M., & R. L. (1988) Biochemistry 27, 1822-1827]. [(OP)2CuI]+ cleavage of a series of oligonucleotide duplexes of the type 5'-CCCTPyPuPyCCCC-3'/3'-GGGAPuPyPuGGGG-5' (Py = pyrimidine; Pu = purine) was examined to determine the effects of purine substituents in the central triplet on specificity. The relative cleavage rates of different PyPuPy triplets in oligomers were similar to those observed for restriction fragments. The undecamer duplex containing the trimer TAT (TTATC) was most preferentially cleaved, predominantly at the central adenosine and the adjacent 3'-thymidine. Duplexes differing from TTATC by a single A.T----G.C transition in the central triplet were cleaved at significantly reduced rates relative to TTATC, the order of preference being TAT greater than TGT greater than TAC greater than CAT. By contrast, duplexes differing from TTATC by a single A.T----I.C transition were cleaved at rates similar to those for TTATC when the transition occurred at the 5'-pyrimidine or central purine [i.e., C(.I)AT and TIT]. A duplex containing the trimer TAC(.I) was cleaved at a reduced rate similar to the duplex containing TAC(.G). The guanine 2-amino group at positions 1 and 2, but not position 3, of a 5'-PyPuPy-3' trimer is therefore implicated as a strong inhibitor of DNA binding by the copper-phenanthroline complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

Tight junctions serve as the rate-limiting barrier to passivemovement of hydrophilic solutes across intestinal epithelia. Afteractivation of Na⁺-glucosecotransport, the permeability of intestinal tight junctions isincreased. Because previous analyses of this physiological tightjunction regulation have been restricted to intact mucosae, dissectionof the mechanisms underlying this process has been limited. Tocharacterize this process, we have developed a reductionist modelconsisting of Caco-2 intestinal epithelial cells transfected with theintestinal Na⁺-glucosecotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstratephysiological Na⁺-glucosecotransport. Activation of SGLT1 results in a 22 ± 5% fall intransepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzinincreases TER by 24 ± 2% (P < 0.001). The increased tight junction permeability is size selective,with increased flux of small nutrient-sized molecules, e.g., mannitol,but not of larger molecules, e.g., inulin. SGLT1-dependent increases intight junction permeability are inhibited by myosin light-chain kinaseinhibitors (20 µM ML-7 or 40 µM ML-9), suggesting that myosinregulatory light-chain (MLC) phosphorylation is involved in tightjunction regulation. Analysis of MLC phosphorylation showed a 2.08-foldincrease after activation of SGLT1 (P < 0.01), which was inhibited by ML-9(P < 0.01). Thus monolayersincubated with glucose and myosin light-chain kinase inhibitors arecomparable to monolayers incubated with phloridzin. ML-9 also inhibitsSGLT1-mediated tight junction regulation in small intestinal mucosa(P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junctionregulation subsequent to SGLT1 activation. The intimate relationshipbetween tight junction regulation and MLC phosphorylation suggests thata critical step in regulation of epithelial tight junction permeabilitymay be myosin ATPase-mediated contraction of the perijunctionalactomyosin ring and subsequent physical tension on the tight junction.

     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells     

Do orthologous gene phylogenies really support tree-thinking?

Background  

Since Darwin's Origin of Species, reconstructing the Tree of Life has been a goal of evolutionists, and tree-thinking has become a major concept of evolutionary biology. Practically, building the Tree of Life has proven to be tedious. Too few morphological characters are useful for conducting conclusive phylogenetic analyses at the highest taxonomic level. Consequently, molecular sequences (genes, proteins, and genomes) likely constitute the only useful characters for constructing a phylogeny of all life. For this reason, tree-makers expect a lot from gene comparisons. The simultaneous study of the largest number of molecular markers possible is sometimes considered to be one of the best solutions in reconstructing the genealogy of organisms. This conclusion is a direct consequence of tree-thinking: if gene inheritance conforms to a tree-like model of evolution, sampling more of these molecules will provide enough phylogenetic signal to build the Tree of Life. The selection of congruent markers is thus a fundamental step in simultaneous analysis of many genes.     

Mobile histone tails in nucleosomes. Assignments of mobile segments and investigations of their role in chromatin folding

The 13C NMR spectrum of isolated nucleosome core particles contains many sharp resonances, including resonances of alpha- and beta-carbons, indicating that certain terminal segments of histones rich in basic residues are highly mobile (Hilliard, R. R., Jr., Smith, R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 5992-5998). Specific histone termini can be removed sequentially from nucleosome core particles by mild treatment with alpha-chymotrypsin or chymotrypsin plus trypsin (Rosenberg, N. L., Smith. R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 12375-12383). Comparisons of the 13C NMR spectra of native and several partially proteolyzed core particles indicated that a minimum of residues 1-20 of H3 and 1-11 and 118-128 of H2a are contained in mobile segments of native cores. H4 did not appear to contribute to the resonances from mobile histone segments, but a possible contribution of H2b residues 1-16 could not be ruled out. The 13C NMR spectra of oligonucleosomes containing and lacking lysine-rich histones (H1, H5) were similar to each other and to that of native nucleosome cores both when the oligonucleosomes were in an extended conformation at low ionic strength and when they were in a more compact conformation at higher ionic strength. This similarity suggests that histones H1 and H5 must be largely immobilized upon chromatin binding and that the segments of core histones that are mobile in isolated nucleosome cores are not strongly bound to adjacent linker regions in intact chromatin, and are not immobilized by compaction to the degree achieved in 50 mM phosphate buffer.     

7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.     

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.