Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.     

Selective passage through the uterotubal junction of sperm from a mixed population produced by chimeras of calmegin-knockout and wild-type male mice

Loss of calmegin, a testis-specific putative chaperone protein of the endoplasmic reticulum, leads to male sterility because the sperm show defects in migration into the oviduct and do not bind to the zona pellucida. To clarify the mechanism of defective migration, XY <--> XY chimeras were produced by aggregating wild-type embryos with embryos of transgenic mice lacking functional calmegin genes and expressing enhanced green fluorescent protein (EGFP) in their acrosomes. Chimeric ejaculates contained wild-type, nonfluorescent sperm as well as sperm with EGFP-tagged acrosomes and the defective calmegin gene. Transgenic, wild-type, and chimeric males were mated to wild-type females; however, only wild-type sperm were ever found within the oviducts. Calmegin-knockout sperm, even when they were combined in chimeric ejaculates with wild-type sperm, remained outside of the uterotubal junction. These findings indicate that the presence of wild-type sperm cannot compensate for the inability of calmegin-knockout sperm to enter the oviduct and that successful ascent into the oviduct depends on the capabilities of individual sperm.     

Full-term development of hamster embryos produced by injection of round spermatids into oocytes

The golden hamster is a mammal in which microinjection of round spermatids into oocytes (ROSI) was first attempted. However, no live ROSI offspring have ever been obtained in this species. This is the first report of live hamster offspring obtained by round spermatid injection. Over 90% of oocytes, injected with round spermatids, were activated without any additional stimulation. The proportion of the oocytes that were fertilized normally and that developed to morulae and blastocysts was higher when the plasma membranes of the spermatids were broken before injection, as compared with when the membranes were left intact. Five percent of 57 ROSI morulae/blastocysts developed into live offspring after transfer to foster mothers.     

Triploid bridge and role of parthenogenesis in the evolution of autopolyploidy

Autopolyploidization is considered to play an important role in plant evolution. In polyploidization, the polyploid evolves from the original diploid cytotype, in which the triploid state is considered to mediate the process (triploid bridge). Nevertheless, the fitness of triploid individuals seems to be too low to facilitate the polyploidization process (triploid block). The evolutionary condition of autopolyploidy was analyzed using a mathematical model focusing on the role of parthenogenesis in triploid and tetraploid individuals. In addition, offspring were assumed to arise by sexual reproduction by conjugations between haploid, diploid, and triploid gametes produced by diploid, tetraploid, and triploid individuals. According to the analysis, even if triploid block suppresses the fitness of sexually produced triploids, the polyploidization process can proceed when parthenogenesis occurs frequently. If only triploids frequently reproduce parthenogenetically, the evolutionary consequences tend to depend on the fitness of the tetraploid individuals. On the basis of a predetermined parameter set, if tetraploid fitness is relatively low, all three ploidies can coexist. Otherwise, tetraploidization occurs. In this case, triploid parthenogenesis promotes not only triploidization but also tetraploidization. However, if both triploids and tetraploids frequently reproduce parthenogenetically, the ploidy levels with the highest fitness are likely to dominate in the population through direct competition among cytotypes.     

Glycosylation site for chondroitin sulfate on the neural part-time proteoglycan, neuroglycan C

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate (CS) proteoglycan that is expressed predominantly in the central nervous system (CNS). NGC dramatically changed its structure from a proteoglycan to a nonproteoglycan form with cerebellar development, whereas a small portion of NGC molecules existed in a nonproteoglycan form in the other areas of the mature CNS, suggesting that the CS glycosylation of NGC is developmentally regulated in the whole CNS. As primary cultured neurons and astrocytes from cerebral cortices expressed NGC in a proteoglycan form and in a nonproteoglycan form, respectively, CS glycosylation seems to be regulated differently depending on cell type. To investigate the glycosylation process, cell lines expressing a proteoglycan form of NGC would be favorable experimental models. When a mouse NGC cDNA was transfected into COS 1, PC12D, and Neuro 2a cells, only Neuro 2a cells, a mouse neuroblastoma cell line, expressed NGC bearing CS chains. In PC12D cells, although three intrinsic CS proteoglycans were detected, exogenously expressed NGC did not bear any short CS chains just like NGC in the mature cerebellum. This suggests that the addition of CS chains to the NGC core protein is regulated in a manner different from that of other CS proteoglycans. As the first step in investigating the CS glycosylation mechanism using Neuro 2a cells, we determined the CS attachment site as Ser-123 on the NGC core protein by site-directed mutagenesis. The CS glycosylation was not necessary for intracellular trafficking of NGC to the cell surface at least in Neuro 2a cells.     

Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation. Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side

The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.     

Archaeal lipids forming a low energy-surface on air-water interface

Archaea or archaebacteria are the microorganism living in extreme environments such as hot springs and salt lakes. The membrane is featured universally by lipids which possess saturated polyisoprenoid chains in the hydrophobic moiety. This paper concerns the surface properties of Langmuir membranes made of archaeal lipid models (AL) bearing a phytanyl group or (3RS, 7R, 11R)-3,7,11,15-tetramethylhexadecyl group. All of the AL provide a Langmuir membrane on an air-water interface with an abnormally low surface tension (32-37 mN/m at 20-70 degrees C), while the conventional lipids having n-alkyl chains give membranes of 54-56 mN/m. The abnormally low energy surface of AL lipids is considered to arise from the bulky and fluid polyisoprenoid chain.     

Identification and biochemical characterization of plant acylamino acid-releasing enzyme

Plant acylamino acid-releasing enzyme (AARE) catalyzing the N-terminal hydrolysis of N(alpha)-acylpeptides to release N(alpha)-acylated amino acids, was biochemically characterized using recombinant and native AAREs. A cDNA encoding a deduced Arabidopsis thaliana AARE (AtAARE) was cloned and sequenced. The deduced amino acid sequence encoded a 764 amino acid protein of 83.9 kDa, which was 31.8% identical with that of rat AARE. In particular, the proposed catalytic residues (Ser, Asp, and His) of AARE, called the "catalytic triad residues, " were completely conserved. Recombinant AtAARE was expressed in Escherichia coli and confirmed to be a functional AARE. Native AAREs were prepared from A. thaliana and cucumber (Cucumis sativus, L.) plants. Both native AAREs were tetrameric proteins of 350 kDa comprising four subunits of 82 kDa, and showed typical enzymological properties of other AAREs, i.e. sensitivity to diisopropyl fluorophosphate, an optimum pH of around 7.0, and an optimum temperature of 37 degrees C. Both the native and recombinant AAREs were immunochemically homologous. Intracelluar fractionation analysis showed that the AARE was mainly present in the stroma of chloroplasts. Native AARE degraded the glycated ribulose-1,5-bisphoshate carboxylase/oxygenase protein but not the native protein. Thus, plant AARE might be involved in not only catalysis of the N-terminal hydrolysis of N(alpha)-acylpeptides but also the elimination of glycated proteins.