Repair of DNA-protein cross-links in mammalian cells

DNA-protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA-protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA-protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA-protein cross-links in duplex DNA (group 4).     

Effects of grazer immigration and nutrient enrichment on an open algae-grazer system

After disturbance, recovery dynamics of local populations depend on arrival rates of immigrants and local growth conditions. We studied the effects of herbivore immigration rates and nutrient enrichment on the dynamics of grazing insect larvae, benthic microalgae, and filamentous macroalgae recovering from low local densities in an open stream system. The two types of algae approximate a trade‐off between capabilities for growing at low resource levels and resisting herbivory. Many microalgae achieve relatively high growth rates at low nutrient levels but are vulnerable to grazers, whereas many macroalgae require high nutrient levels for growth but become increasingly defended with filament growth. We hypothesized that macroalgae should benefit more strongly than microalgae from increasing nutrient levels and decreasing grazer immigration rates, because both conditions increase macroalgal chances to grow into a size refuge from herbivory. We created a gradient of nutrient concentrations and manipulated drift immigration rates of macroinvertebrates. Macro‐ and microalgal biomass and the relative contribution of macroalgae to total algal biomass increased with increasing nutrient enrichment and decreased with increasing grazer immigration. Grazer densities responded positively to nutrient enrichment. The densities of large baetids responded positively to higher immigration rates of large baetids, whereas small baetids and chironomid larvae showed the opposite response. Per capita emigration of small baetids decreased with increasing algal biomass. The data suggest that large baetids negatively affected algal biomass and that small baetid and chironomid densities tracked resource levels set by nutrient enrichment and large baetids. Our experiments highlight the prospects of integrating disturbance with nutrient supply, immigration rates and local trophic interactions (determining recovery trajectories) into conceptual models of open system dynamics. We suggest that recovery trajectories towards micro‐ or macroalgal dominated states may depend on the spatial scale of disturbance relative to the movement ranges of migrating grazers and to nutrient supply.     

Effects of nucleotide sequence alignment on phylogeny estimation: a case study of 18S rDNAs of apicomplexa

The reconstruction of phylogenetic history is predicated on being able to accurately establish hypotheses of character homology, which involves sequence alignment for studies based on molecular sequence data. In an empirical study investigating nucleotide sequence alignment, we inferred phylogenetic trees for 43 species of the Apicomplexa and 3 of Dinozoa based on complete small-subunit rDNA sequences, using six different multiple-alignment procedures: manual alignment based on the secondary structure of the 18S rRNA molecule, and automated similarity-based alignment algorithms using the PileUp, ClustalW, TreeAlign, MALIGN, and SAM computer programs. Trees were constructed using neighboring-joining, weighted-parsimony, and maximum- likelihood methods. All of the multiple sequence alignment procedures yielded the same basic structure for the estimate of the phylogenetic relationship among the taxa, which presumably represents the underlying phylogenetic signal. However, the placement of many of the taxa was sensitive to the alignment procedure used; and the different alignments produced trees that were on average more dissimilar from each other than did the different tree-building methods used. The multiple alignments from the different procedures varied greatly in length, but aligned sequence length was not a good predictor of the similarity of the resulting phylogenetic trees. We also systematically varied the gap weights (the relative cost of inserting a new gap into a sequence or extending an already-existing gap) for the ClustalW program, and this produced alignments that were at least as different from each other as those produced by the different alignment algorithms. Furthermore, there was no combination of gap weights that produced the same tree as that from the structure alignment, in spite of the fact that many of the alignments were similar in length to the structure alignment. We also investigated the phylogenetic information content of the helical and nonhelical regions of the rDNA, and conclude that the helical regions are the most informative. We therefore conclude that many of the literature disagreements concerning the phylogeny of the Apicomplexa are probably based on differences in sequence alignment strategies rather than differences in data or tree-building methods.      

Mycoparasites effect on reproductive ability of Sclerotinia sclerotiorum sclerotia.

The ability to parasitise Sclerotinia sclerotiorum and the effect on apothecia production was evaluated for the following antagonists: Trichoderma harzianum; Trichoderma koningii; Gliocladium roseum and Chaetomium globosum. Plastic trays were filled with of steam-sterilized soil. Each one of them was infested with sclerotia of S. sclerotiorum and the culture of the antagonists. The trays were kept in a greenhouse and after 30, 60 and 90 days, evaluations were made. The rates of carpogenic germination, myceliogenic germination, mycoparasitism and destruction were evaluated. To assess carpogenic germination, the sclerotia were put in a growth chamber over moistened filter paper at 20 -/+ 2 degrees C and 12 light hours. The rates of myceliogenic germination and mycoparasitism were evaluated on Petri dishes with 2% APD. Antagonists effect on carpogenic germination was observed one month after the start of the assay. In the evaluation made at 60 and 90 days, T. harzianum; T. koningii and G. roseum kept inhibitory properties. Such inhibition was not observed in the trays containing C. globosum. In the evaluations made at 30 days, mycoparasitism rate was high in the trays with T. harzianum; T. koningii and G. roseum. G. roseum and T. harzianum destroy S. sclerotiorum sclerotia.     

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross- hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.      

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

Protease production by Leuconostoc oenos strains isolated from wine

Two separate, extracellular proteolytic activities were demonstrated in four strains of Leuconostoc oenos isolated from Argentinian wines. The first took place in the early growth phase and the other had its maximum at the end of growth. The two proteolytic enzymes had different pH and temperature optima. Divalent metal ions had different effects not only on each L. oenos strain but also on each of the two proteases from each strain.     

The requirement of IHF protein for extrachromosomal replication of the Escherichia coli oriC in a mutant deficient in DNA polymerase I activity

It is shown here that plasmids containing the replication origin of Escherichia coli (oriC) cannot replicate in an extrachromosomal state in E. coli cells with the polA1hip3 double mutation. This E. coli mutant is deficient in the polymerizing function of DNA polymerase I (Pol I) and is unable to produce functional IHF protein. The inability of the oriC minichromosomes to replicate in the absence of IHF is dependent on the absence of Pol I; cells with the polA+himA- or polA+hip- mutation, which are deficient in the alpha and beta subunits of the IHF heterodimer, respectively, can support replication of the oriC replicons. We propose that IHF-deficient cells utilize an alternative pathway of the DNA replication in which Pol I is required. In vitro DNA binding assays revealed that the IHF binding site resides between the oriC coordinates 110 and 122 and is adjacent to the DnaA "box" 1. Within the area protected by IHF we found at least 1 out of 11 GATC methylation sites present in oriC. The consequences of lack of IHF protein binding to the oriC and the indirect effects of the IHF deficiency on the oriC replication are discussed.     

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