Structural analysis of Arabidopsis thaliana chromosome 5. VIII. Sequence features of the regions of 1,081,958 bp covered by seventeen physically assigned P1 and TAC clones.

A total of 17 Pl and TAC clones each representing an assigned region of chromosome 5 were isolated from P1 and TAC genomic libraries of Arabidopsis thaliana Columbia, and their nucleotide sequences were determined. The length of the clones sequenced in this study summed up to 1,081,958 bp. As we have previously reported the sequence of 9,072,622 bp by analysis of 125 P1 and TAC clones, the total length of the sequences of chromosome 5 determined so far is now 10,154,580 bp. The sequences were subjected to similarity search against protein and EST databases and analysis with computer programs for gene modeling. As a consequence, a total of 253 potential protein-coding genes with known or predicted functions were identified. The positions of exons which do not show apparent similarity to known genes were also assigned using computer programs for exon prediction. The average density of the genes identified in this study was 1 gene per 4277 bp. Introns were observed in 74% of the potential protein genes, and the average number per gene and the average length of the introns were 4.3 and 168 bp, respectively. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Effects of Some Calcium-Related Agents on the Protoplast Transfection of Lactobacillus casei with Phage PL-1 DNA

To clarify the mechanism of Ca²⁺involvement in the DNA transfer through cell membrane, we studied the effects of Ca²⁺-chelator, Ca²⁺-ionophore, and Ca²⁺-channel blocker on the protoplast transfection of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in the presence of Ca²⁺. Ca²⁺-chelators, citrate, EDTA, and dipicolinic acid, inhibited the transfection probably by compensating the effect of Ca²⁺. Ca²⁺-ionophores, A23187 and N,N,N′,N′-tetracyclohexyl-3-oxapentanediamide, which were expected to accelerate transfection by introducing Ca²⁺ into cells, inhibited the transfection. This fact indicated the absence of correlation between the entry of Ca²⁺ and the transport of DNA into protoplasts. Verapamil, which blocks voltage-dependent Ca²⁺-channel besides β-adrenergic receptor, inhibited the transfection with little effect on the survival of the protoplasts. Both flunarizine and vinpocetine, voltage-dependent Ca²⁺-channel blockers, did not show the selective toxicity. D-α-Aminoadipic acid, a glutamate receptor-operated Ca²⁺-channel blocker, had no effect. Propranolol, which blocks β-adrenergic receptor as does verapamil, inhibited the transfection without severely damaging the protoplasts. These results suggested that a kind of receptor-operated Ca²⁺-channel was involved in the transport of PL-1 phage DNA into the cells and that the cell membrane might have a receptor structure somewhat similar to the β-adrenergic receptor found in mammalian cells. Received: 6 May 1996 / Accepted: 10 June 1996     

Ethanol production by encapsulated and immobilized yeast

Summary Immobilized yeast was encapsulated with cell-free calciumalginate gel by two-step preparation procedure. The volume of coated film decreased with increasing cell concentration. The encapsulation did not affect ethanol production and could prevent cell leakage from the gels.     

Establishment of inbred rabbit strains

Two inbred rabbit strains, JWY-NIBS and NWY-NIBS, were established by full-sib mating in the Nippon Institute for Biological Science, Laboratory Animal Research Station. The origin, breeding history, and biological characteristics of the two inbred strains are as follows. 1. JWY-NIBS: Origin: Japanese white rabbits which had been originally kept at a farm near mount Takao, then transferred to another farm in Fuchu and maintained as a closed colony for a long time. Date of the first full-sib mating: April, 1964. Date of the 20th full-sib mating: June, 1981. Age of maturity: 7-month-old in females, 7.5-month-old in males. Body weight at maturity: 2.8-3.0 kg in females, 2.8-2.9 kg in males. Marker genes: Hemopexin; HxS. Esterase; Est-1s. alpha-Protein; F type. 2. NWY-NIBS: Origin: A strain III of New zealand white rabbits imported from the Jakson Laboratory in USA. Date of the first full-sib mating: November, 1967. Date of the 20th full-sib mating: July, 1982. Age of maturity: 7.5-month-old in females, 8-month-old in males. Body weight at maturity: 2.8-3.0 kg in females, 2.9-3.1 kg in males. Marker genes: Hemopexin; HxF. Esterase; Est-1S. Est-2f. alpha-Protein; S type.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.     

Frequent Detection of Hepatitis C Virus Subtype 3a (HCV-3a) Isolates in Thailand by PCR Using Subtype-Specific Primers

By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or ?2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and ?1b) and 2 (HCV-2a and ?2b).