Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

“绿色革命”新进展:赤霉素与氮营养双重调控的表观修饰助力水稻高产高效育种

以半矮秆育种为代表的“绿色革命”极大地提高了作物产量,但也带来氮营养利用效率降低的严重问题。“绿色革命”主要基于调控赤霉素的代谢和信号转导而实现。前期的研究发现,赤霉素信号转导关键因子DELLA蛋白通过调控GRF4而负调控氮素的吸收利用,为半矮秆品系氮利用效率低的问题提供了解决方案。最近的一项研究进一步揭示了GA信号途径与氮响应交叉互作的新机制。该研究发现水稻(Oryza sativa)NGR5是氮素调控分蘖数目的一个关键基因,其表达受氮诱导。通过招募PRC2,NGR5对D14和OsSPL14等分蘖抑制基因所在位点进行H3K27me3甲基化修饰,从而抑制其表达。而在半矮秆背景下超表达NGR5可以提高低氮水平下的水稻产量。NGR5同时也被发现为赤霉素受体GID1的一个新靶标,受到其负调控。该研究发现了调控赤霉素信号通路的新机制,并对高产高效的新一代“绿色革命”育种实践具有重要启示。     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

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Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden‐headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden‐headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm–egg‐binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short‐term measure for the conservation of golden‐headed lion tamarins.     

Reproductive morphology of the male Tuatara,Sphenodon punctatus

Over the past decade, studies on reproductive morphology in the Squamata (snakes and lizards) have expanded tremendously. With the accumulation of these studies and revisions of the terminology based on structural similarities and differences, it is imperative to review the work on tuataras to determine whether the structural organization fits the revised terminology of vertebrates. We investigated the morphology of the male reproductive system in the Tuatara, Sphenodon punctatus (Rhynchocephalia), the sister taxon to the Squamata. Previous studies on the Tuatara used a nomenclature for the testicular ducts different from the current terminology for amniotes. The reproductive system in the Tuatara is consistent with reports in the Squamata. Two rete testis tubules exit the testis within a connective tissue sheath similar to that shown in other squamate species and the protherian Echidna. Each rete testis divides into multiple ductuli efferentes that fuse with the epididymis. The epididymis transitions into the ductus deferens where the sperm become more concentrated into spherical bundles. The ductus deferens enters the cloacal urodeum separately from the ureter. An ampulla ureter or ampulla urogenital papilla was not observed, which differs from previous studies of lepidosaurians. Furthermore, a sexual segment of the kidney (SSK) was not observed, consistent with previous studies on the Tuatara.     

Single-channel activity in sea urchin sperm revealed by the patch-clamp technique

Ionic fluxes are deeply involved in the response of spermatozoa to the egg. Using the patch-clamp technique, we show for the first time single ion channel activity in sea urchin spermatozoa and spermatozoa heads. Due to their small size gigaseals were obtained in suspended cells by applying suction through the pipette. The rate of gigaseal formation was very low and improved to 6% (n = 1145) when flagella were detached from sperm. Current-voltage curves created from single-channel events showed conductances of approx. 65 and 170 pS, suggesting the presence of two types of channels. At least one appears to be a K⁺ channel.     

Statistical evidence for remnants of the primordial code in the acceptor stem of prokaryotic transfer RNA

Summary The specificity of interaction of amino acids with triplets in the acceptor helix stem of tRNA was investigated by means of a statistical analysis of 1400 tRNA sequences. The imprint of a prototypic genetic code at position 3–5 of the acceptor helix was detected, but only for those major amino acids, glycine, alanine, aspartic acid, and valine, that are formed by spark discharges of simple gases in the laboratory. Although remnants of the code at position 3–5 are typical for tRNAs of archaebacteria, eubacteria, and chloroplasts, eukaryotes do not seem to contain this code, and mitochondria take up an intermediary position. A duplication mechanism for the transposition of the original 3–5 code toward its present position in the anticodon stern of tRNA is proposed. From this viewpoint, the mode of evolution of mRNA and functional ribosomes becomes more understandable.Offprint requests to: W. Moller     

A new procedure for rapidly scoring acrosome reactions of human sperm

Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm.     

A Dimeric Structural Scaffold for PRC2-PCL Targeting to CpG Island Chromatin

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