Male pipefish prefer dominant over attractive females

Animals may obtain information guiding their choice betweenpotential partners from observing competitive interactionsand displays between them, or from displays directed at thechoosing individual. In the sex-role reversed pipefish Syngnathustyphle females display a temporary ornament (a color pattern)to other females as well as to males. We have previously shown that display of female ornaments per se is attractive to males.Here we show that information from competitive displays canoverride such direct attraction displays as signals in thepartner choice process. In a mate choice experiment, an enclosedmale could choose between two females. On the first experimentalday, females could interact freely, while on the second daythey were isolated from each other. When female-female competitionwas allowed, the ornament display was directed more to theother female than to the male: Time competing, rather thantime courting the male, correlated with ornament display duration.However, ornament display under competition and ornament displayin the absence of competition did not correlate significantly.In fact, females competing more intensively on day one displayedthe ornament less on day two. Furthermore, the ornament displayduring the first, but not the second, day predicted male matechoice on the second day. Thus, males remembered previous informationfrom competitive displays and used it rather than immediateinformation from displays in the absence of female-female competition.We suggest that competitive displays more reliably signal female quality as compared to noncompetitive ones, and that males benefitfrom mating with dominant females.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Female sand gobies prefer good fathers over dominant males

Female preference for males successful in male-male competition is generally assumed to result in mating with high quality males. Here I report results from an experiment disentangling the effects of intra- and intersexual selection in the sand goby, Pomatoschistus minutus, a marine fish that exhibits paternal care. I show that large males are successful in male–male competition, but contrary to what one would expect, dominants are not preferred by females and are not better at taking care of the eggs. Female preference, however, correlated with the subsequent hatching success of the eggs. Thus, female choice selects for good parenting. Hence, direct benefits in the form of superior paternal care can explain female choice in this species, supporting a good parent process of sexual selection. However, choosing on the outcome of male–male competition does not enable females to mate with the ''best'' males.     

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.     

Heterochromatin protein 1 binds to nucleosomes and DNA in vitro

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. The purpose of this study was to test whether HP1 can bind directly to nucleosomes. HP1 binds nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. We show that histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues had no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures.     

The characterisation of 1SF monomer nucleosomes from hen oviduct and the partial characterisation of a third HMG14/17-like in such nucleosomes.

Nucleosomes released from oviduct nuclei during brief micrococcal nuclease digestions are enriched in transcribed sequences (bloom K.S. and Anderson, J.N. (1978) Cell, 15, 141-150). Such nucleosomes released into this 1Sf supernatant fraction are enriched in proteins HMG14, 17 and a third lower molecular weight protein which we show in this paper to be related to HMG14 and 17. This protein, which we call HMGY, runs as a doublet on polyacrylamide gels. A similar doublet is present in smaller quantities in chicken erythrocyte nuclei. Monomer nucleosomes in the 1SF supernatant have been separated by polyacrylamide gel electrophoresis into two main bands. The slower moving band contains the three HMG proteins HMG14, 17 and Y but lacks histone H1.     

Pyrrolysine analogs as substrates for bacterial pyrrolysyl-tRNA synthetase in vitro and in vivo

Pyrrolysine-tRNA(Pyl) complex is produced by pyrrolysyl-tRNA synthetase (PylRS). In this study, we investigated the substrate specificity of Desulfitobacterium hafnience PylRS. PylRS incorporated various L-lysine derivatives into tRNA(Pyl) in vitro. In addition, the PylRS/tRNA(Pyl) pair introduced these lysine derivatives into the recombinant protein by the Escherichia coli expression system, indicating that this PylRS/tRNA(Pyl) pair can be used in protein engineering technology.     

Native HMGB1 protein inhibits repair of cisplatin-damaged nucleosomes in vitro

The high mobility group box (HMGB) 1 protein, one of the most abundant nuclear non-histone proteins has been known for its inhibitory effect on repair of DNA damaged by the antitumor drug cisplatin. Here, we report the first results that link HMGB1 to repair of cisplatin-treated DNA at nucleosome level. Experiments were carried out with three types of reconstituted nucleosomes strongly positioned on the damaged DNA: linker DNA containing nucleosomes (centrally and end-positioned) and core particles. The highest repair synthesis was registered with end-positioned nucleosomes, two and three times more efficient than that with centrally positioned nucleosomes and core particles, respectively. HMGB1 inhibited repair of linker DNA containing nucleosomes more efficiently than that of core particles. Just the opposite was the effect of the in vivo acetylated HMGB1: stronger repair inhibition was obtained with core particles. No inhibition was observed with HMGB1 lacking the acidic tail. Binding of HMGB1 proteins to different nucleosomes was also analysed. HMGB1 bound preferentially to damage nucleosomes containing linker DNA, while the binding of the acetylated protein was linker independent. We show that both the repair of cisplatin-damaged nucleosomes and its inhibition by HMGB1 are nucleosome position-dependent events which are accomplished via the acidic tail and modulated by acetylation.     

16S rRNA Methyltransferases as Novel Drug Targets Against Tuberculosis

The Protein Journal - Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (M.tb) whose natural history traces back to 70,000 years. TB remains a major...     

Acetoacetate and beta-D-hydroxybutyrate as energy substrates during early bovine embryo development in vitro

We examined the effects of acetoacetate and other metabolic products of fatty acid oxidation on early bovine embryo development. In vitro produced bovine zygotes were cultured in modified-synthetic oviduct fluid medium supplemented with acetoacetate, acetoacetate derivatives, acetyl CoA precursors and lithium chloride. Acetoacetate and all acetoacetate derivatives, with the exception of the ethyl ester, supported in vitro development up to the hatched blastocyst stage at rates similar to that of controls supplemented with lactate/pyruvate. The optimal concentration of acetoacetate in supporting embryo development was 3.6 mM; addition of 1.8 and 3.6 mM lithium chloride did not significantly affect embryo development, while 7.2 mM was inhibitory. Hatched blastocysts cultured with 3.6 mM acetoacetate contained a similar number of cells as the lactate/pyruvate control group. It can be concluded that in vitro produced bovine embryos can develop using ketone bodies as energy substrates, which could be derived in vivo from endogenous lipids.     

T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.     

The yeast GAL1-10 UAS region readily accepts nucleosomes in vitro

To test if the absence of nucleosomes on the UAS region of the yeast GAL1-10 genes in vivo could be due to a low inherent affinity of this DNA for histones, DNA fragments containing the UAS and various amounts of flanking DNA were reconstituted into chromatin. Restriction enzyme and DNase I digestion analyses show that DNA in the UAS becomes protected against digestion in the reconstitutes. Thus, nucleosomes can assemble on the UAS region in vitro. The level of protection of the UAS and of the flanking DNA regions is comparable and remains so at various levels of nucleosome loading, suggesting that the UAS DNA has no tendency to exclude nucleosomes. In fact, DNase I results suggest that the UAS elements themselves preferentially bind histones.