Biomedical subjects

R Sasada

Publications and source records attributed to R Sasada.

43 records · Page 3Linked to original sources

PubMed1985

Establishment of hybridomas secreting monoclonal antibodies against C epsilon 2 and C epsilon 4 domains of human IgE.

Three kinds of hybridomas secreting monoclonal antibodies (MAbs) against the epsilon chain of human IgE were constructed by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with human IgE purified from the culture supernatant of U-266 cells. These MAbs were used effectively for the purification and determination of human IgE. The recognition site in the IgE molecule of each antibody was examined by using various epsilon chain fragment peptides produced in Escherichia coli. From these experiments, it was suggested that one recognized C epsilon 2 and the second C epsilon 4. The third did not recognize the C epsilon 1-C epsilon 4 domains of the recombinant epsilon chain from E. coli, although it bound to the epsilon chain of natural human IgE.

Animals↗

PubMed1983

Direct expression of hepatitis B surface antigen gene in E. coli.

A 809 bp Sau 3A - Hpa I fragment containing a complete HBsAg gene and fragments 744 bp Hinc II - Hpa I and 712 bp Xba I - Hpa I containing a truncated HBsAg gene lacking the sequence encoding the NH2-terminal hydrophobic domain were prepared from a composite plasmid pHBV933 containing the 3.2 kb Eco RI DNA fragment of the entire HBV/adw genome and inserted into an expression vector pTRP801 to give plasmids pTRP SS-6, pTRP SS-39, and pTRP SS-50, respectively. The growth of a recombinant having pTRP SS-6 was greatly inhibited and the transformant expressed a low level of HBsAg, which is reactive to human anti-HBsAg antibody. Interestingly, the growth of transformants harbouring pTRP SS-39 and pTRP SS-50 was not inhibited and these transformants expressed a considerable level of the HBsAg. Minicells harbouring pTRP SS-6, pTRP SS-39, and pTRP SS-50 formed specific polypeptides of about 24 K, 23 K, and 22 K daltons, respectively.

Antigen-Antibody Complex↗

PubMed1983

Expression of human immunoglobulin E epsilon chain cDNA in E. coli.

Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.

Base Sequence↗

PubMed1983

The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw.

The complete nucleotide sequences of two different subtypes (adr and adw) of hepatitis B virus (HBV) DNA cloned in E. coli were determined. The sequence of the viral genome of the adr clone was 3188 nucleotides long, and that of the adw clone was 3200 nucleotides long. The adr and adw clones differed from the reported cloned ayw HBV DNA (3182 nucleotides long) in 11.2% and 10.0% of nucleotides, respectively. Heterogeneity of the HBV genome in the clones with the same subtype was observed.

Amino Acid Sequence↗

PubMed1983

Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.

Amino Acid Sequence↗

PubMed1979

Spontaneous production of a C-type RNA virus in a cell line derived from rat glioma.

The spontaneous production of a rat C-type RNA virus (ACV) in a cultured cell line (AC cells) established from a chemically induced rat glioma was studied. The characteristics of ACV were: morphology typical of C-type RNA virus; buoyant density of 1.15 g/ml in a sucrose density gradient; RNA directed DNA polymerase activity; viral core with a density of 1.28 to 1.30 g/ml; 70S RNA with dimer structure; and structural protein composed of mainly four polypeptides. Kinetical analysis of DNA-DNA hybridization revealed that DNA sequences homologous to DNA transcripts of RNA of ACV were present in rat cells. RNA directed DNA polymerase of ACV partially cross-reacted with antiserum to the polymerase of Rauscher murine leukemia virus. These data suggest that ACV is an endogenous C-type RNA virus of rat origin.

Animals↗

PubMed1978

Biochemical studies on bovine adenovirus type 3. IV. Transformation by viral DNA and DNA fragments.

By the calcium technique, intact DNA of bovine adenovirus type 3 (BAV3) was found to transform A31 cells, a clone of BALB/3T3. Transforming activity was resistant to RNase and Pronase but sensitive to DNase. The efficiency of transformation was approximately 5 to 10 foci per mug of DNA. Attempts were also made to test for transforming activity of BAV3 DNA fragments prepared with restriction endonucleases EcoRI and HindIII. The activity was found to associate exclusively with the EcoRI D fragment mapped in the region of 3.6 and 19.7 units (molecular weight, 3.9 x 10(6)). No transformation could be obtained with three HindIII fragments, J, E, and B, located at the left-hand end of the BAV3 genome. However, the enzymatic joining of J and E fragments (0 to 11.9 map units) with a ligase restored the transforming activity. These results suggest that all the genetic information of BAV3 required for transformation is located in the region between 3.6 and 11.9 units on the viral genome. Some properties of A31 cells transformed by BAV3 DNA EcoRI D fragment (TrD) and the ligated DNA of HindIII J and E fragments (TrJE), as well as those transformed by whole BAV3 DNA (Tr), were examined. As compared to untransformed A31 cells, all the transformed cell lines tested showed rapid growth, high saturation densities, and anchorage-independent growth. Moreover, they contained BAV3-specific T antigen and induced tumors in adult nude and BALB/c mice. These properties of Tr, TrD, and TrJE lines were similar to those of BAV3-transformed cells.

Adenoviridae↗