Biomedical subjects

M Wakiyama

Publications and source records attributed to M Wakiyama.

24 records · Page 2Linked to original sources

PubMed1997

Expression of Xenopus laevis translation initiation factor 4E (eIF-4E) by baculovirus-insect cell system.

A gene encoding Xenopus laevis eIF-4E was cloned into a transfer vector, and its gene expression was attempted in cells of E. coli, yeast and insect. Effective expression of the active eIF-4E was achieved in the soluble fraction of the insect cell Sf9, which was infected with the recombinant baculovirus. Overexpression of the eIF-4E protein caused remarkable change in the shape of the cells.

Animals↗

PubMed1997

Characteristic distribution of bases and codons around the initiation and termination codons in whole reading frames in bacteria and yeast genomes.

Recently the complete nucleotide sequence of the entire genome was determined for yeast and a few kinds of bacterium. To see characteristic features of base sequence in the cistron (actually the open reading frame, ORF) and in the regions around a cistron (ORF), the biases of appearance frequency of bases from the base ratio were studied statistically. In the regions before the base biases were observed. The characteristic base distribution patterns were similar to all the cases of bacteria, but different from yeast. The base biases are reflected on the appearance frequency of amino acids near the N-termini and C-termini of proteins. The characteristic biases found in the amino acid sequence of the N-terminal part of bacteria proteins are different from that in yeast proteins.

Bacillus subtilis↗

PubMed1995

mRNA encoding the translation initiation factor eIF-4E is expressed early in Xenopus embryogenesis.

The translation initiation factor eIF-4E plays an important role in regulating the overall rate of translation in eukaryotic cells. To investigate the expression of eIF-4E itself, we characterized the eIF-4E mRNA expressed in Xenopus embryos. 5'-RACE was performed to determine the 5'-end of the mRNA and the result predicts isoforms differing at the amino-terminal end. Expression of the eIF-4E mRNA in Xenopus oocytes and embryos was examined by RT-PCR. Xenopus eIF-4E mRNA is produced during oogenesis and persists during the early stages of embryogenesis as a maternal mRNA.

Activin Receptors↗

PubMed1994

High-level expression of porcine muscle adenylate kinase in Escherichia coli: effects of the copy number of the gene and the translational initiation signals.

Porcine muscle adenylate kinase (ADK) was overproduced in Escherichia coli using the expression plasmid with double A-T-G codon at the translational starting site and the Shine-Dalgarno (SD) sequence 10 bp apart from the first A-T-G. We used the expression vectors pKK223-3 and pMK2. pMK2 is about 10-20 times larger in copy number than pK223-3. For both vectors, duplication of A-T-G was effective and the quantity of the expressed ADK from the double A-T-G plasmid was 2 approximately 4-fold more than that achieved when only one A-T-G was present. The amount of the produced ADK was maximum in the case of using pMK2 with double A-T-G. The overproduced ADK formed inclusion bodies in E. coli. It was solubilized in 6 M guanidine hydrochloride and refolded. Through two steps of column chromatography, ADK was purified. It has the same amino acid composition and grossly the same activity as that reported by Schirmer et al. (1970). Its amino acid sequence of the NH2-terminal region was identical with that deduced from the cDNA sequence including the NH2-terminal methionine.

Adenylate Kinase↗

PubMed1993

Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence.

The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5' leader region of brome mosaic virus (BMV) RNA4 and the goat pre-alpha-lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.

Animals↗

PubMed1992

Relationship between the 5'-leader sequence of mRNAs and the translation efficiency in a eukaryotic in vitro system.

To examine the effect of the 5'-leader region of mRNAs in regulating translation efficiency, chimeric mRNAs which have various sequences of the 5'-leader were synthesized by the SP6 in vitro transcription system, and translated in the wheat germ in vitro translation system. Translation efficiency was affected by the length of the 5'-leader and the number of the codon A-U-G at the translation initiation site.

Animals↗