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T Uetsuki

Publications and source records attributed to T Uetsuki.

27 records · Page 2Linked to original sources
PubMed1991

A stable cellular marker for the analysis of mouse chimeras: the bacterial chloramphenicol acetyltransferase gene driven by the human elongation factor 1 alpha promoter.

We have developed a method of marking of mouse cells by means of transfection of a foreign gene. The transgene chosen here was the plasmid pEF321CAT which contains the bacterial chloramphenicol acetyl transferase (CAT) gene linked to the promoter region of the human polypeptide chain elongation factor 1 alpha (hEF1 alpha) gene. Evaluation of the plasmid pEF321CAT as a cellular marker for mouse cells involved intensive examination of a transgenic mouse carrying pEF321CAT. The CAT gene was expressed in all tissues examined, demonstrating that the hEF1 alpha promoter was active in a wide range of mouse cells. The plasmid itself did not exert any harmful effect on the normal development of mice, and the CAT activity was immunohistologically detectable on sectioned tissues by the use of anti-CAT serum. When the plasmid was transferred into embryonal carcinoma (EC) cells and embryonic stem (ES) cells, the CAT gene was also found to be expressed constantly irrespective of their differentiation. These results demonstrated that the plasmid pEF321CAT can be used as a reliable and feasible cellular marker that would distinguish unequivocally the cells of each of genotype in chimeric tissues.

Aging↗
PubMed1990

Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system.

We have characterized the promoter region of the human elongation factor 1 alpha-encoding gene (EF-1 alpha) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1 alpha gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1 alpha gene: (i) ligated to the end of the TATA box (pEF220-CAT); (ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and (iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.

Animals↗
PubMed1990

Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes.

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.

Animals↗
PubMed1989

Isolation and characterization of the human chromosomal gene for polypeptide chain elongation factor-1 alpha.

The cDNA for human elongation factor-1 alpha was isolated from a cDNA library of human fibroblast cells. Using the cDNA as a probe, a number of chromosomal genes encoding the human elongation factor-1 alpha were isolated. Characterization of the clones by restriction enzyme mapping and nucleotide sequence analysis has revealed that only one of them is an active gene, whereas all of the other genes are processed pseudogenes. The active gene consists of 8 exons and 7 introns spanning about 3.5 kilobases, and the sequence of its exons is completely identical to that of the human elongation factor-1 alpha cDNA. The first non-coding exon of 33 base pairs is separated by a 943-base pair intron from the coding exons. The primer extension of human elongation factor-1 alpha mRNA has indicated that the transcription of human elongation factor-1 alpha gene starts from a C residue, and a "TATA" box was found 24 base pairs upstream of the initiation site. Three and five Sp1 binding sites are present on the 5'-flanking region and the 1st intron, respectively. Furthermore, one Ap-1 binding site is located in the 1st intron. By using nuclear extracts from HeLa cells, the promoter of human elongation factor-1 alpha gene could stimulate in vitro transcription better than the adenovirus major late promoter.

Amino Acid Sequence↗