Macrocyclic piperazinones as potent dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I

A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for the binding mode of compound 3o in FPTase is proposed.     

Extreme difference in rate of mitochondrial and nuclear DNA evolution in a large ectotherm, Galápagos tortoises

We sequenced approximately 4.5 kb of mtDNA from 161 individuals representing 11 named taxa of giant Galápagos tortoises (Geochelone nigra) and about 4 kb of non-coding nuclear DNA from fewer individuals of these same 11 taxa. In comparing mtDNA and nucDNA divergences, only silent substitutions (introns, ITS, mtDNA control region, and synonymous substitutions in coding sequences) were considered. mtDNA divergence was about 30 times greater than that for nucDNA. This rate discrepancy for mtDNA and nucDNA is the greatest yet documented and is particularly surprising for large ectothermic animals that are thought to have relatively low rates of mtDNA evolution. This observation may be due to the somewhat unusual reproductive biology and biogeographic history of these organisms. The implication is that the ratio of effective population size of nucDNA/mtDNA is much greater than the usually assumed four. The nearly neutral theory of molecular evolution predicts this would lead to a greater difference between rates of evolution.     

Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

Metal ion requirements for structure and catalysis of an RNA ligase ribozyme

The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.     

Characterization of Trichodesmium spp. by genetic techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus TRICHODESMIUM: The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of KATAGNYMENE: Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.     

The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective

Background

Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species.

Results

We identified 85 known and eight novel families of transposable element varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence. More than two-thirds of the transposable elements are partial. The density of transposable elements increases an average of 4.7 times in the centromere-proximal regions of each of the major chromosome arms. We found that transposable elements are preferentially found outside genes; only 436 of 1,572 transposable elements are contained within the 61.4 Mb of sequence that is annotated as being transcribed. A large proportion of transposable elements is found nested within other elements of the same or different classes. Lastly, an analysis of structural variation from different families reveals distinct patterns of deletion for elements belonging to different classes.

Conclusions

This analysis represents an initial characterization of the transposable elements in the Release 3 euchromatic genomic sequence of D. melanogaster for which comparison to the transposable elements of other organisms can begin to be made. These data have been made available on the Berkeley Drosophila Genome Project website for future analyses.     

Dinitrogen fixation in the world's oceans

The surface water of themarine environment has traditionally beenviewed as a nitrogen (N) limited habitat, andthis has guided the development of conceptualbiogeochemical models focusing largely on thereservoir of nitrate as the critical source ofN to sustain primary productivity. However,selected groups of Bacteria, includingcyanobacteria, and Archaea canutilize dinitrogen (N₂) as an alternativeN source. In the marine environment, thesemicroorganisms can have profound effects on netcommunity production processes and can impactthe coupling of C-N-P cycles as well as the netoceanic sequestration of atmospheric carbondioxide. As one component of an integrated Nitrogen Transport and Transformations project, we have begun to re-assess ourunderstanding of (1) the biotic sources andrates of N₂ fixation in the world'soceans, (2) the major controls on rates ofoceanic N₂ fixation, (3) the significanceof this N₂ fixation for the global carboncycle and (4) the role of human activities inthe alteration of oceanic N₂ fixation. Preliminary results indicate that rates ofN₂ fixation, especially in subtropical andtropical open ocean habitats, have a major rolein the global marine N budget. Iron (Fe)bioavailability appears to be an importantcontrol and is, therefore, critical inextrapolation to global rates of N₂fixation. Anthropogenic perturbations mayalter N₂ fixation in coastal environmentsthrough habitat destruction and eutrophication,and open ocean N₂ fixation may be enhancedby warming and increased stratification of theupper water column. Global anthropogenic andclimatic changes may also affect N₂fixation rates, for example by altering dustinputs (i.e. Fe) or by expansion ofsubtropical boundaries. Some recent estimatesof global ocean N₂ fixation are in therange of 100–200 Tg N (1–2 × 10¹⁴ g N)yr^–1, but have large uncertainties. Theseestimates are nearly an order of magnitudegreater than historical, pre-1980 estimates,but approach modern estimates of oceanicdenitrification.