Phenol-treatment and a homologous pairing-assay.

Homologous pairing is a key step in homologous genetic recombination. In the early stage of trials for the identification of homologous pairing-promoting proteins from a fission yeast, Schizosaccharomyces pombe, we treated DNA products with phenol in the presence of a salt for the removal of tightly bound proteins from DNA before the assay, but we found that this treatment caused very efficient protein-independent double-strand formation from complementary single-stranded DNAs. Using an assay including the phenol treatment, we detected another species of apparent homologous pairing-promoting proteins in the nuclei, in addition to a homologous pairing-promoting protein consisting of three components which we reported previously. However, studies involving the use of an assay without the phenol-treatments revealed that the second one was not really a homologous pairing-protein. Thus, the protein-independent double-strand formation by phenol-treatment in the presence of a salt could cause the erroneous identification of homologous pairing-promoting proteins.     

Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium

Explants excised from strawberry (Fragaria x ananassa Duch.) plantlets were cultured in vitro for 21 days on half-strength MS (Murashige & Skoog 1962) basal liquid medium with 20 g l^-1 sucrose and without sugar in the vessels capped with gas permeable microporous polypropylene film. The experiments were conducted under CO₂ nonenriched (350–450 mol mol^-1 in the culture room) and CO₂ enriched (2,000 mol mol^-1 during the photoperiod in the culture room) conditions with a PPF (photosynthetic photon flux) of 200 mol m^-2 s^-1. The CO₂ concentration in the vessels decreased to approximately 200 mol mol^-1 during the photoperiod on day 21 under CO₂ nonenriched conditions. The fresh and dry weight, net photosynthetic rate (NPR) per plantlet, NPR per g leaf fresh weight, NPR per g leaf dry weight, the number of unfolded leaves, and ion uptake of PO₄ ^3-, NO₃ ^-, Ca²⁺, Mg²⁺ and K⁺ on day 21 were the greatest under photoautotrophic (no sugar in the medium) and CO₂ enriched conditions. The residual percent of PO₄ ^3- was 3% on day 21 under photoautotrophic and CO₂ enriched conditions.Abbreviations MS Murashige & Skoog (1962) basal medium composition - NPR net photosynthetic rate - PPF photosynthetic photon flux     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum acstivum L.) mature embryos

We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of thegusA (GUS) gene was detected when dry embryos were vortexed for 10–30 min in a SCF-DNA solution containing 90–120 g/l of sucrose. Up to 100 (on average 20–40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUS-expressing sectors one month after treatment.     

Effect of penicillin G on the electroporation of Rhodococcus rhodochrous CF222

M. SUNAIRI, N. IWABUCHI, K. MURAKAMI, F. WATANABE, Y. OGAWA, H. MUROOKA AND M. NAKAJIMA. 1996. Suitable conditions for the introduction of bacteriophage DNA into cells of Rhodococcus rhodochrous CF222 by electroporation were established, and penicillin G was found to enhance the transfection frequency. When conditions optimal for the parental strain were applied to its colony-morphological mutants, different transfection frequencies were observed. Penicillin G enhanced the transfection frequency of smooth and mucoidal mutants but not of rough mutants.     

Somatic mosaicism of expanded CAG repeats in brains of patients with dentatorubral-pallidoluysian atrophy: cellular population-dependent dynamics of mitotic instability.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disease caused by unstable expansion of a CAG repeat in the DRPLA gene. We performed detailed quantitative analysis of the size and the size distribution (range) of the expanded CAG repeats in various regions of the CNS of eight autopsied patients with DRPLA. Expanded alleles (AE) showed considerable variations in size, as well as in range, depending on the region of the CNS, whereas normal alleles did not show such variations, which indicates the occurrence of somatic mosaicism of AE in the CNS. The AE in the cerebellar cortex were consistently smaller by two to five repeat units than those in the cerebellar white matter. Moreover, the AE in the cerebral cortex were smaller by one to four repeat units than those in the cerebral white matter. These results suggest that the smaller AE in the cerebellar and cerebral cortices represent those of neuronal cells. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter showed considerable variation ranging from 9 to 23 repeat units, whereas those in the cerebellar cortex showed little variance and were approximately 7 repeat units. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter were much broader in patients with higher ages at death than they were in patients with lower ages at death, raising the possibility that the range of AE increases with time, as the result of mitotic instability of AE.