Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.     

An increase in circulating mast cell colony-forming cells in asthma

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.     

Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism

Antioxidant activities of 15 purified bilberry anthocyanins together with pelargonidin 3-O-beta-D-glucopyranoside and 4'-O-methyl delphinidin 3-O-beta-D-glucopyranoside (MDp 3-glc), the major metabolite of delphinidin 3-O-beta-D-glucopyranoside (Dp 3-glc), were evaluated in order to study the structure-antioxidant activity relationship and any synergism among them in the mixture. Both aglycone structure and the attached sugar moiety affected the O*2- and ONOO- -scavenging activities, although the effect of the attached sugar moiety was smaller than that of the aglycone structure. The potency of activity toward the superoxide radical was in the following order: delphinidin > petunidin > malvidin =approximately cyanidin>(+)-catechin > peonidin > pelargonidin. The activity toward ONOO- was: delphinidin > cyanidin =approximately petunidin > malvidin =approximately (+)-catechin > peonidin > pelargonidin. It was confirmed that methylation of 4'-OH markedly reduced the antioxidant activity of anthocyanin. Further, it was revealed that synergism occurred in both - and ONOO- -scavenging activities among the anthocyanins in the mixture.     

DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline

Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.     

Cloning and expression of the chicken immunoglobulin joining (J)-chain cDNA

 The J chain is a component of polymeric immunoglobulin (Ig) molecules and may play an important role in their polymerization and the transport of polymeric Ig across epithelial cells. In this study, the primary structure of the chicken J chain was determined by sequencing cDNA clones. The cDNA had an open reading frame of 476 nucleotides encoding a putative protein of 158 amino acid residues including the signal sequence. The 3′ untranslated region consisted of 1216 nucleotides and a poly(A) tail. The deduced amino acid sequence of the chicken J chain had a high degree of homology to that of human, cow, rabbit, mouse, frog, and earthworm, with eight conserved Cys residues identical to the mammalian J chains. Northern blot hybridization performed with total RNA from various chicken tissues revealed high levels of J-chain mRNA expression in spleen, intestine, Harderian gland, and bursa of Fabricius, and low levels in the thymus. The J chain was expressed in the bursa as early as day 15 of embryogenesis. These data indicated that the chicken J-chain gene displays a high degree of homology with that of other species, and is expressed at an early stage of development of the chicken immune system. Received: 21 May 1999 / Revised: 30 August 1999     

Amino acid sequences of ovomucoid third domains from 27 additional species of birds

Ovomucoids consist of a single polypeptide chain which is composed of three tandem Kazal domains. Each Kazal domain is an actual or putative protein inhibitor of serine proteinases. Ovomucoid third domains were already isolated and sequenced from 126 species of birds (Laskowskiet al., 1987, 1990). This paper adds 27 new species. A number of generalizations are made on the basis of sequences from 153 species. The residues that are in contact with the enzyme in enzyme-inhibitor complexes are strikingly hypervariable. While the primary specificity residue,P ₁, is the most variable; substitutions occur predominantly among aliphatic, hydrophobic residues. Consensus sequences for an avian ovomucoid third domain, for a b-type Kazal domain (i.e., a COOH terminal domain of multidomain inhibitors) and for a general Kazal domain are given. Finally, the individual new sequences are briefly discussed.     

Responses of Trachelospermum asiaticum (Apocynaceae) seedlings to growth in a light intensity gradient

We recorded the growth of 24 seedlings of Trachelospermum asiaticum, a root climber, placed between a light source and a wall. Shoot length of seedlings planted at the brightest points nearly matched the distance the shoot tips moved toward the wall surface. In contrast, although the seedlings planted at the darkest points did elongate, the tips moved an average distance of only 0.4 cm. Creeping shoots of T. asiaticum planted in brighter environments exhibit negative phototropism, which encourages them to grow rapidly toward dark places, allowing them to reach supporting hosts faster than those that germinate close to the host.