Temperature gradient DNA-probe column chromatography: as tools for detection and purification of particular DNAs and RNAs

We have developed the temperature-gradient DNA-probe column chromatography as the new method for detecting and purifying particular DNAs or RNAs accurately. The method has high discrimination resolution, and can detect a single-base change in sample base sequences. The method also has high performances in purification of particular DNAs or RNAs, which has been demonstrated by purification of bovine mitochondria serine t-RNA. None of other method can succeed in complete purification of this t-RNA molecule. The present method can be applied not only to molecular biology as basic tools for purifying and picking-up particular sequences but also to the medical field as diagnostic tools.     

Molecular cloning of cDNA coding for human preprourokinase

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, fractionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.     

A new hereditary single band variant of the Gc system

Summary A new single band variant (Gc Ar) or the Gc subtypes not identical with the known Gc variants has been detected in the plasma of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by a single band which has a similar isoelectric point to the Gc 1C2 anodal band. It is well known that the single band Gc phenotypes remain unaltered after neuraminidase treatment. Nevertheless, the new single band variant (Gc Ar) is altered after neuraminidase treatment as is Gc 2A3. After neuraminidase treatment, the Gc Ar band is affected and moved to the nearby position of the Gc 2 band. Investigation of the proband's family shows that the variant occurs combined with the common alleles Gc 1F, Gc 1S and that it has an autosomal dominant inheritance.     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Reconstitution mechanism of nucleosome core particles mediated by poly(l-glutamic acid)

Poly(l-glutamic acid) has been reported to mediate in vitro nucleosome assembly (Stein, A., Whitlock, J.P., Jr. and Bina, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5000–5004). To study the reaction mechanism, we have reconstituted nucleosome core particles from chicken erythrocyte core DNA and core histones in the presence of poly(l-glutamic acid) and analyzed the assembly products by polyacrylamide gel electrophoresis. Poly(l-glutamic acid), which binds and forms a large complex with core histones, is replaced with core DNA in the reconstitution process. When histone-poly(l-glutamic acid) complex and core DNA are mixed with a histone:DNA ratio of 1.0, the yield of core particles increases by prolonged reconstitution time. Two phases with a distinct time range appear in the process. In the fast phase within 30 min, 60% of the DNA is involved in products containing histones: reconstituted core particles, a larger nucleoprotein complex and aggregation. In the second phase, the remaining DNA and the DNA in the aggregation decrease, and the core particles increase slowly. The yield of core particles is approx. 60% after 24 h. The slow phase is not observed by reconstitution with a histone:DNA ratio of 2.0 in the initial mixture. The reaction scheme of the assembly process derived from these data is given. Based on the in vitro reaction scheme, the possible role of in vivo ‘nucleosome assembly factors’ is also discussed.     

Special somatic spine synapses in the ciliary ganglion of the chick

Summary Somatic spine synapses modified with postsynaptic electron opaque materials were found in the axo-somatic ciliary ganglion synapse of the chick.A part of the postsynaptic cell body protrudes into the presynaptic calyciform ending as a somatic spine with about 1 in length and 0.15 in diameter, and forms the so-called synaptic complex with presynaptic process. Moreover, conspicuous electron opaque materials can be seen in the central axis of the spine, except for its end portion. Sometimes, these opaque materials are seen as arrayed dots.The morphological characteristics of the somatic spine synapses in this study are quite similar to that found in the habenula and interpeduncular nuclei of the cat (Milhaud and Pappas, 1966). the biological significance of which is obscure at present.This work was supported in part by grant from the Education Ministry of Japan.