Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Probing of contacts between <Emphasis Type="Italic">Eco</Emphasis>RII DNA methyltransferase and DNA with the use of substrate analogs and molecular modeling

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

Parallel self-associated structures formed by T,C-rich sequences at acidic pH

Oligonucleotides of nonregular heteropyrimidine sequences incorporating or not incorporating purine residues 5'-d(ACTCCCTTCTCCTCTCTA), 5'-d(ACTCCCTGGTCCTCTCTA), 5'-d(TCTCTCCTGGTCCCTCC), and 5'-d(TCTCTCCTCTTCCCTCC) can form self-associated parallel-stranded (ps) structures at pH 4-5.5. The ps structures were identified by studying at neutral and acidic pH UV melting transitions, FTIR spectra, and fluorescence of pyrene-labeled oligonucleotides as well as by chemical joining of 5'-phosphorylated oligonucleotides. A gel electrophoresis run for oligonucleotides 5'-d(TCTCTCCTCTTCCCTCC) and 5'-d(ACTCCCTTCTCCTCTCTA) has shown the formation of homoduplexes at low DNA strand concentrations. Ps structures are held by C-C(+) base pairs and have N- and S-types of sugar puckering as detected by FTIR spectroscopy in the millimolar concentration range. Guanine inserts as well as thymine and purine inserts into an oligomeric cytosine sequence make the formation of the tetraplex i-motif unfavorable. MvaI restriction endonuclease, which recognizes the CCT/AGG sequence in DNA, does not cleave parallel pseudosubstrates.     

Probing of contacts between EcoRII DNA methyltransferase and DNA using substrate analogs and molecular modeling

To determine the molecular mechanism of DNA recognition and catalysis by EcoRII DNA-methyltransferase (M.EcoRII) binding and methylation by the enzyme of 14-mer substrate analogs containing 2-aminopurine or 1',2'-dideoxy-D-ribofuranose in the M.EcoRII recognition site have been studied. Efficiencies of methylation and DNA binding affinities depend on the location of modified nucleoside residues within the M.EcoRII recognition site. A structural model of M.EcoRII in complex with substrate DNA and cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was built using the previously solved structures of Hhal and HaeIII DNA-methyltransferases as templates. The model was constructed according to the recently developed "Frankenstein's monster" approach. Based on the model, amino acid residues taking part in interactions with DNA were predicted. Besides, based on both theoretical and experimental data obtained the groups of atoms of the heterocyclic bases within the M.EcoRII recognition site presumably involved in interaction with the enzyme were proposed.     

Experimental studies of the behavior of the ion plasma component during disruptive instability in a tokamak

Results are presented from experimental studies of the behavior of the plasma ion component during disruptive instability in the TVD and DAMAVAND tokamaks. It is shown that the ion temperature increases during a major disruption by a factor of 1.5–2. The ions are accelerated predominantly across the magnetic field near the rational magnetic surfaces. Results on the ion acceleration along the magnetic field indicate that disruptions are accompanied by the generation of longitudinal electric fields that are aligned in opposite directions at the plasma periphery and near the plasma axis.     

DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction- modification enzymes.

To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regioselective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-beta-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the Eco RII and Mva I R-M enzymes that contained a galactose or periodate-oxidized galactose residue as single substituents either in the center of the Eco RII (Mva I) recognition site or in the flanking nucleotide sequence. The dependence of binding, cleavage and methylation of these substrate analogs on the modified sugar location in the duplex was determined. Cross-linking of the reagents to the enzymes under different conditions was examined. M. Eco RII covalent attachment to periodate-oxidized substrate analogs proceeded in a specific way and to a large extent depended on the location of the reactive dialdehyde group in the substrate. The yield of covalent attachment to a DNA duplex with a dialdehyde group in the flanking sequence with Eco RII or Mva I methylases was 9-20% and did not exceed 4% for R. Eco RII.     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays