Biomedical subjects

M Yokoyama-Kobayashi

Publications and source records attributed to M Yokoyama-Kobayashi.

6 recordsLinked to original sources

PubMed1999

Selection of cDNAs encoding putative type II membrane proteins on the cell surface from a human full-length cDNA bank.

We have developed a simple method to test whether a hydrophobic segment near the N-terminus of a protein functions as a type II signal anchor (SA) in which the N-terminus faces the cytoplasm. A cDNA fragment containing the putative SA sequence of a target clone was fused in-frame to the 5' end of a cDNA fragment encoding the protease domain of urokinase-type plasminogen activator (u-PA). The resulting fused gene was expressed in COS7 cells. Fibrinolytic activity on the cell surface was measured by placing a fibrin sheet in contact with the transfected COS7 cells after removing the medium. When the cDNA fragment encoded a SA, the fibrin sheet was lysed by the u-PA expressed on the cell surface. The fibrinolytic activity was not detected in the culture medium, suggesting that the u-PA remains on the cell surface anchored via the SA in the membrane without being cleaved by signal peptidase. This fibrin sheet method was successfully applied to select five novel cDNA clones encoding putative type II membrane proteins from a human full-length cDNA bank.

Animals↗

PubMed1997

Human cDNA encoding a novel TGF-beta superfamily protein highly expressed in placenta.

Recently, we developed a simple method for detecting a secretory signal sequence encoded by a cDNA fragment. In this study, we used this method to select cDNA clones encoding secretory proteins from a human full-length cDNA library. Full-sequencing analysis of the candidate clones revealed that one clone encoded a novel TGF-beta superfamily protein. The clone encodes a protein of 308 amino acids of which the C-terminal region shows a characteristic feature of TGF-beta superfamily proteins: seven conserved cysteine residues at the C-terminal preceded by a putative processing site composed of a basic amino acid repeat. The corresponding transcripts are highly expressed in the placenta, so the novel protein may play an important role in reproduction.

Amino Acid Sequence↗

PubMed1995

A signal sequence detection system using secreted protease activity as an indicator.

We have developed a novel expression vector, pSSD1, to detect a cDNA fragment encoding a secretory signal sequence (Ss). This vector carries a cDNA fragment encoding the protease domain of human urokinase-type plasminogen activator (u-PA) as a reporter gene under the SV40 early promoter. We inserted cDNA fragments encoding various Ss between the promoter and the reporter gene of pSSD1. These plasmids were introduced into monkey COS7 cells to produce chimeric proteins. Only when the Ss was intact, was fibrinolytic activity detected in the culture medium of the transfected cells. Thus, this system may be useful for detecting the cDNAs encoding secreted proteins or type-I membrane proteins.

Amino Acid Sequence↗

PubMed1994

Construction of a human full-length cDNA bank.

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKA1, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.

Animals↗

PubMed1994

Recombinant f1 phage-mediated transfection of mammalian cells using lipopolyamine technique.

Recombinant f1 phages carrying a shuttle vector pKA1M for expression of blasticidin S deaminase were introduced into monkey COS7 cells by mixing with dioctadecylamidoglycylspermine (DOGS). Blasticidin S selection resulted in the detectable growth of resistant colonies within a week. The transfection efficiency depended on the amounts of the phage and DOGS, their ratio, and the time during which the cells were incubated with the phage/DOGS mixture. This method requires only several microliters of an Escherichia coli culture medium containing recombinant f1 phage particles and is applicable to various cell lines including mouse NIH/3T3, chinese hamster CHO-K1, and human HT-1080.

3T3 Cells↗

PubMed1993

Recombinant f1 phage particles can transfect monkey COS-7 cells by DEAE dextran method.

Recombinant f1 phage particles containing cDNA of the human urokinase type-plasminogen activator (u-PA) under the control of the simian virus 40 (SV40) early promoter were used for transfection of monkey COS-7 cells using a DEAE dextran method. The fibrinolytic activity of u-PA was detected in the culture medium of the 10(5) cells transfected with the phage particles containing single-stranded (ss) DNA of more than 0.2 ng. This finding will lead us to develop a simple and efficient method for expression cloning using mammalian cells.

Animals↗