Tadayoshi Shiba

Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector

To examine the validity of the strategy to express a foreign gene as a fusion protein with the coat protein (CP) of tobacco mosaic virus (TMV), we have constructed ENK RNA by using an in vitro transcription system of TMV RNA. ENK RNA differs from TMV RNA only in that ENK RNA carries an additional sequence coding for Leu‐enkephalin (Tyr‐Gly‐Gly‐Phe‐Leu) (Enk) with a preceding in‐frame methionine just before the termination codon of CP gene. In protoplasts inoculated with ENK RNA, CP+Enk fusion protein accumulated as the major protein.

The nucleotide sequences of copia and copia-related RNA in Drosophila virus-like particles

We have shown previously that Drosophila cells contain virus-like particles (VLPs) containing 5-kilobase (kb) RNA that hybridizes to a transposable element, termed copia. We have suggested that VLPs and copia are derivatives of viral particles and proviral forms, respectively, of ‘copia’ retrovirus, a putative Drosophila retrovirus1. To further clarify the relationship between copia and copia-related RNA in VLPs (VLP H-RNA), we determined and compared their nucleotide sequences. VLP H-RNA was found to be an unspliced, genome-sized transcript of copia, and, like retroviral genome RNA, VLP H-RNA is terminally redundant with termini localized in the long terminal repeats (LTRs) of copia. VLP H-RNA contains two long open reading frames (ORFs), one of which includes the coding sequence for a predominant VLP protein of relative molecular mass (Mr) 31,000 (31K). Here we show that, in contrast to 17.6 ORF2 (ref. 2), ORFs of copia have no extensive amino-acid sequence homology to the RT region2 of the reverse transcriptase of retrovirus in vertebrates. Because of a one-base insertion/deletion, the two ORFs in VLP H-RNA are fused and become a single, longer ORF in a genomic copia.

Metal-inducible activities of metallothionein promoters in fish cells and fry

Activities of trout metallothionein-A promoter and mouse metallothionein-I promoter in fish cells and fry were examined using the chloramphenicol acetyltransferase (CAT) gene as a reporter. Transfection and transient CAT assays on a trout liver cell line indicated that both promoters were inducible by metals, including zinc. In vivo activities of both promoters were examined using microinjection system into medaka eggs. Some of the hatched fry were exposed to zinc and their CAT activities were assayed. Both promoters revealed considerable activities after exposure to zinc while only weak activities were detected in unexposed fry.

Constitutive and inducible expression of a transgene directed by heterologous promoters in a trout liver cell line.

Activities of heterologous promoters and enhancers in cultured rainbow trout liver cells were examined employing the bacterial chloramphenicol acetyltransferase gene as the reporter. SV40 promoter-enhancer and Rous sarcoma virus long terminal repeat directed constitutive expression at high levels. Moloney murine leukemia virus long terminal repeat and SV40 promoter combined with Adenovirus type 2 enhancer were also constitutively expressed. Drosophila Hsp70 promoter was activated when the transformed cells were cultured at 25 degrees C, a higher temperature than the temperature normally used, in faithful response to heat shock.

Structure of Drosophila virilis glycerol-3-phosphate dehydrogenase gene and a comparison with the Drosophila melanogaster gene

The complete glycerol-3-phosphate dehydrogenase gene of Drosophila virilis isolated by screening with alpha GPDHM cDNA of the adult fly was sequenced. The gene contains eight exons spread over a total of approximate 8 kb DNA. Its exon/intron organization is identical to that of D. melanogaster. A single transcription initiation site was determined by primer extension. The stop codons are located at the 3 end of each of the exons 6 to 8. TATA and CAAT boxes are present upstream of the transcriptional start site. Adult alpha GPDH protein is encoded by exons 1 to 6 and exon 8. Comparison of the sequence with that of D. melanogaster showed that the homology of the nucleotide sequence of the coding region is 85% and that the homology of the amino acid sequence is 98%. On the contrary, the non-coding region is quite different in length and nucleotide sequence.

Identification of an unusual structure in the Drosophila melanogaster transposable element copia: evidence for copia transposition through an RNA intermediate

The Drosophila melanogaster transposable element copia is usually 5 kb long with long terminal repeats (LTRs), and its major transcripts are a full-length 5-kb RNA and a 2-kb RNA. We have previously shown that the 2-kb RNA is generated through splicing. Here, we have cloned a genomic intronless copia using an oligodeoxyribonucleotide probe which is specific for the junction of the two exons. The unusual copia is bounded by two LTRs and lacks precisely the intron of the 2-kb copia RNA. Identification of genomic intronless copia strongly suggests that copia transposes through an RNA intermediate. Moreover, we have found that copia virus-like particles (VLPs), in which reverse transcription of copia RNA seems likely to occur, packages the spliced copia RNA much less efficiently than the full-length copia RNA. This result leads to the suggestion that much lower copy number of genomic intronless copia, as compared with that of normal copia, may be responsible for the inefficient packaging of the spliced copia RNA into the VLP.

Production of a unique multi-lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor

Drosophila retrotransposon copia produces virus‐like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi‐lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization otthe major copia VLP protein, and Furthermore, would provide important information concerning the mechanism of copia VLPs formation.

Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.